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Objective: To investigate Th2 differentiation features of Mycobacterium tuberculosis heat resistant antigens (MTB-HAg)-activated human γδT cells and the regulation of transcription factor T-box expression in T cells (T-bet) and GATA-binding protein 3 (GATA-3) on differentiation., Methods: Peripheral blood mononuclear cells (PBMCs) were stimulated with MTB-HAg to generate MTB-HAg-activated T cells (MTBAT) and expanded in the neutral condition or Th2 polarizing condition. After restimulation for 6 hours with phorbol myristate acetate (PMA, 10 ng/mL), ionomycin (500 ng/mL) and monensin (2.5 μmol/L), intracellular cytokines (IFN-γ, IL-4) of γδT cells and αβT cells among MTBAT were detected by four-color fluorescence mAb staining combined with flow cytometry. The highly purified γδT cells and CD4⁺ T cells were sorted by flow cytometer from MTBAT that were cultured in neutral and Th2 polarizing conditions for 28 days. The expressions of T-bet and GATA-3 mRNA in purified γδT cells and CD4⁺ T cells were determined by reverse transcription PCR (RT-PCR) technique., Results: γδT cells among MTBAT cultured in the neutral or Th2 polarizing condition predominantly produced IFN-γ, whereas the percentage of IFN-γ⁺ αβT cells significantly decreased in the Th2 polarizing condition as the culture time went by. Compared with the neutral condition, Th0 type (IFN-γ⁺ IL-4⁺) γδT cells significantly increased, and Th2 type (IFN-γ⁻ IL-4⁺) αβT cells also significantly increased in the Th2 polarizing condition. RT-PCR showed that mRNA expression of T-bet was still at a high level in γδT cells that were expanded in the Th2 polarizing condition, but at a low level in Th2 polarized CD4⁺ T cells. Moreover, the mRNA expressions of GATA-3 in both Th2 polarized γδT cells and CD4⁺T cells were up-regulated., Conclusion: In the Th2 polarizing condition, the majority of γδT cells in MTBAT still remained Th1 profile, whereas the portion of γδT cells differentiated into Th0 type cells. Both transcription factor T-bet and GATA-3 failed to display a fully cross-regulation function in Th2 polarized γδT cells.

Our previous study shows that conventional protein kinases C (cPKCs) are key signaling mediators that are activated by extracellular inhibitory molecules. Inhibition of cPKC by intrathecal infusion of a cPKC inhibitor, GÖ6976, into the site of dorsal hemisection (DH) induces regeneration of lesioned dorsal column sensory, but not corticospinal tract (CST), axons. Here, we investigated whether a direct cortical delivery of GÖ6976 into the soma of corticospinal neurons promotes regeneration of CST and the recovery of forelimb function in rats with cervical spinal cord injuries. We report that cortical delivery of GÖ6976 reduced injury-induced activation of conventional PKCα and PKCβ1 in CST neurons, promoted regeneration of CST axons through and beyond a cervical DH at C4, formed new synapses on target neurons caudal to the injury, and enhanced forelimb functional recovery in adult rats. When combined with lenti-Chondroitinase ABC treatment, cortical administration of GÖ6976 promoted even greater CST axonal regeneration and recovery of forelimb function. Thus, this study has demonstrated a novel strategy that can promote anatomical regeneration of damaged CST axons and partial recovery of forelimb function. Importantly, such an effect is critically dependent on the efficient blockage of injury-induced PKC activation in the soma of layer V CST neurons., (© The Author 2013. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Background: Esophageal squamous cell carcinoma (ESCC) is one of the most lethal malignancies with a 5-year survival rate less than 15%. Understanding of the molecular mechanisms involved in the pathogenesis of ESCC becomes critical to develop more effective treatments., Methods: Mcl-1 expression was measured by reverse transcription (RT)-PCR and Western blotting. Human Mcl-1 promoter activity was evaluated by reporter gene assay. The interactions between DNA and transcription factors were confirmed by electrophoretic mobility shift assay (EMSA) in vitro and by chromatin immunoprecipitation (ChIP) assay in cells., Results: Four human ESCC cell lines, TE-1, Eca109, KYSE150 and KYSE510, are revealed increased levels of Mcl-1 mRNA and protein compare with HaCaT, an immortal non-tumorigenic cell line. Results of reporter gene assays demonstrate that human Mcl-1 promoter activity is decreased by mutation of kappaB binding site, specific NF-kappaB inhibitor Bay11-7082 or dominant inhibitory molecule DNMIkappaBalpha in TE-1 and KYSE150 cell lines. Mcl-1 protein level is also attenuated by Bay11-7082 treatment or co-transfection of DNMIkappaBalpha in TE-1 and KYSE150 cells. EMSA results indicate that NF-kappaB subunits p50 and p65 bind to human Mcl-1-kappaB probe in vitro. ChIP assay further confirm p50 and p65 directly bind to human Mcl-1 promoter in intact cells, by which regulates Mcl-1 expression and contributes to the viability of TE-1 cells., Conclusions: Our data provided evidence that one of the mechanisms of Mcl-1 expression in human ESCC is regulated by the activation of NF-kappaB signaling. The newly identified mechanism might provide a scientific basis for developing effective approaches to treatment human ESCC.

Objective: To explore new hallmarks affecting the prognosis of patients with laryngeal squamous cell carcinoma (LSCC) via investigating the expression of CyclinE and p27 in LSCC tissues., Method: The expression of CyclinE and p27 was detected via Elivision immunohistochemical staining in 160 LSCC tissues and 20 normal laryngeal tissues (NLT). The relationship between CyclinE/ p27 and LSCC/ NLT was analyzed via Log-rank analysis. The relationship of CyclinE and p27 protein was statistically analyzed by spearman correlation analysis. The relationship between CyclinE/p27 and clinical-pathology-factors of patients with LSCC, such as age, gender, tumor site, diameter, differentiation, lymph node metastasis and PTNM stage were analyzed by Chi-square test. The relationship between clinical-pathology-factors, CyclinE, p27 and overall survival time of patients with LSCC was analyzed via Cox multiplicity and Kaplan-Meier survival analysis. A significant difference was recognized by P<0.05., Result: In LSCC the positive rates of CyclinE and p27 protein was 62.50% and 41.25% respectively (P<0.05). In NLT the positive rates of CyclinE and p27 protein was 35% and 70% respectively (P<0.05). The expression of CyclinE or p27 protein was closely correlated with lymph node metastasis, PTNM stage of patients with LSCC (P<0.05). The expression of CyclinE and p27 had no significant correlations with patients' gender, age and tumor site, diameter differentiation (P>0.05 for all). A negative correlation was found between the expression of CyclinE and p27 protein, r= -0.767(P<0.05). Kaplan-Meier survival analysis showed that the overall survival rate of patients with LSCC was 36.9% (P<0.05). The 5-year survival rate in positive group of CyclinE was 8%, in negative group was 80% (P<0.05). On the contrary, the 5-year survival rate of patients with LSCC in positive group of p27 protein was 77.27%, the rate was 5.32% in negative group (P<0.05). Cox multivariate regression analysis revealed that lymph node metastasis, PTNM stage, CyclinE and p27 were independent risk factors of prognosis for patients with LSCC., Conclusion: It is the molecular basis underlying the development and invasion/ metastasis of LSCC that activation of CyclinE gene accompanying inactivation of p27 gene. It is very important of co-detecting CyclinE and p27 protein to predict the prognosis of patients with LSCC.

Background: Activation of the renin-angiotensin system (RAS) plays a critical role in the pathophysiology of myocardial infarction (MI) and the development of heart failure. Both angiotensin-converting enzyme 2 (ACE2) and insulin/insulin receptor substrate-1 (IRS-1) show cardioprotective effects after acute MI. The Arg972 IRS-1 polymorphism is associated with diminished activity of insulin. In the present study, we explored the association among Arg972 IRS-1, acute MI, and serum levels of ACE2., Methods: A total of 711 subjects, including 351 subjects with first-time acute MI and 360 subjects without a history of MI were genotyped for Arg972 IRS-1 polymorphism. Serum levels of ACE2 and MI severity scores were determined. Primary human cardiomyocytes with overexpression of wild type IRS-1 or Arg972 IRS-1 or knockdown of endogenous IRS-1 were exposed to normoxia and hypoxia, and the expression levels of ACE2 were determined., Results: The serum ACE2 level was significantly increased in acute MI patients compared with that of non-MI controls. Compared with wild type IRS-1 carriers, Arg972 IRS-1 carriers exhibited decreased serum ACE2 levels and increased MI severity scores after MI. Our in vitro data demonstrate that impairment of insulin/IRS-1/PI3K signaling by overexpression of Arg972-IRS-1, knockdown of endogenous IRS-1, or PI3K inhibitor can abolish hypoxia-induced IRS-1-associated PI3K activity and ACE2 expression in human cardiomyocytes, which suggests a causal relationship between Arg972-IRS-1 and decreased serum ACE2 levels in acute MI patients. Our in vitro data also indicate that insulin/IRS-1/PI3K signaling is required for ACE2 expression in cardiomyocytes, and that hypoxia can enhance the induction effect of insulin/IRS-1/PI3K signaling on ACE2 expression in cardiomyocytes., Conclusions: This study provides the first evidence of crosstalk between insulin/IRS-1/PI3K signaling and RAS after acute MI, thereby adding fresh insights into the pathophysiology and treatment of acute MI.

The barrier function of the esophageal epithelium is a major defense against gastroesophageal reflux disease. Previous studies have shown that reflux damage is reflected in a decrease in transepithelial electrical resistance associated with tight junction alterations in the esophageal epithelium. To develop novel therapies, it is critical to understand the molecular mechanisms whereby contact with a refluxate impairs esophageal barrier function. In this study, surgical models of duodenal and mixed reflux were developed in mice. Mouse esophageal epithelium was analyzed by gene microarray. Gene set enrichment analysis showed upregulation of inflammation-related gene sets and the NF-κB pathway due to reflux. Significance analysis of microarrays revealed upregulation of NF-κB target genes. Overexpression of NF-κB subunits (p50 and p65) and NF-κB target genes (matrix metalloproteinases-3 and -9, IL-1β, IL-6, and IL-8) confirmed activation of the NF-κB pathway in the esophageal epithelium. In addition, real-time PCR, Western blotting, and immunohistochemical staining also showed downregulation and mislocalization of claudins-1 and -4. In a second animal experiment, treatment with an NF-κB inhibitor, BAY 11-7085 (20 mg·kg⁻¹·day⁻¹ ip for 10 days), counteracted the effects of duodenal and mixed reflux on epithelial resistance and NF-κB-regulated cytokines. We conclude that gastroesophageal reflux activates the NF-κB pathway and impairs esophageal barrier function in mice and that targeting the NF-κB pathway may strengthen esophageal barrier function against reflux.

To summarize the case of combined heart-lung transplantation for a patient who survived for 8.5 years. On September 20, 2003, at Second Xiangya Hospital of Central South University, homologous heartlung transplantation was performed on a male patient who was diagnosed with cardiopulmonary failure secondary to congenital ventricular septal defect with severe pulmonary hypertension. Heart-lung allograft was preserved with 1500 mL modified St.Thomas solution and 3000 mL modified LPD solution. Postoperative immunosuppressive therapies included: methylprednisolone and human anti-lymphocyte globulin protein in the induction period; and combination of ciclosporin A, CellCept and prednisolone in the stable period. In 2007, the treatment was changed to CellCept mg, twice a day+FK506 4 mg, twice a day. The patient lived 8.5 years of normal life with cardiac function of NYHA I-II. Echocardiogram showed left ventricular ejection fraction of 61% to 74%. Heart-lung transplantation proved reliable therapy modality for terminal cardiopulmonary failure. Excellent donor organ preservation and proper perioperative treatment are key factors for long-term survival after heart-lung transplantation.

T-helper (Th) 17 and the more recently identified Th22 cells are of great importance in host defense against pathogens, but can also be responsible for chronic inflammatory disorders. However, the roles of the two cell subsets in syphilis remain elusive. In this study, we show that the frequencies of Th17 and Th22 cells are significantly increased in the peripheral blood of patients with secondary syphilis (SS). A significant positive correlation is observed between Th17 and Th22 cells, whereas a negative correlation exists between Th17 and Th1 cells. Moreover, the frequency of Th17 cells has a significant positive correlation with the plasma interleukin 6 (IL-6) or IL-1β levels, and the frequency of Th22 cells is positively correlated with the IL-6 or IL-23 levels. Finally, the elevated frequencies of Th17 and Th22 cells are positively associated with plasma C-reactive protein levels. Our results suggest that Th17 and Th22 cells may be implicated in the pathogenesis of the SS., (© 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Proliferation of pulmonary artery smooth muscle cells (PASMC) is an important contributor to the progress of pulmonary arterial hypertension (PAH). Anti-inflammatory therapies may have therapeutic applicability for PAH. Resveratrol (RES) has prominent anti-inflammatory effects in vitro, but in vivo its beneficial effects are limited by short systemic half life and poor lipotrophy. A derivative of RES, trimethoxystilbene (TMS), has higher lipotropy and extended half life compared with RES, and can potentially overcome the limitations of RES. In the present study, we studied the effects of TMS and RES on proliferation and apoptosis of PASMC stimulated with Tumor Necrosis Factor-α. The effects on PASMC proliferation were quantified by MTT, while apoptosis was assessed by flow cytometry (Annexin V/propidium iodide assay). We observed strong inhibitory effects of TMS on the growth of PASMC, and these effects were 20 times more potent than those of RES. We further documented induction of apoptosis in PASMC treated with TMS, again, to a higher degree than with RES. In conclusion, TMS is more potent than RES in the inhibition of proliferation and induction of apoptosis of PASMC, demonstrating its potentially beneficial role for treating PAH.

Background: Importin13 (IPO13) is a novel potential marker of corneal epithelial progenitor cells. We investigated the expression and localization of IPO13 in endometrial, endometriotic and endometrial carcinoma tissue., Material/methods: IPO13 expression in endometrial, endometriotic and endometrial carcinoma tissue was examined by immunohistochemistry, qPCR and Western blot., Results: Immunohistochemistry studies showed that IPO13 protein was expressed mainly in cytoplasm of glandular epithelial cell and stromal cells. The rate of importin13-positive cells in proliferative phase endometrium was higher (by about 6-fold) than that in secretory endometrium (P<0.05) and the rate of importin13-positive cells in endometriosis and endometrial carcinoma was higher than that in normal secretory phase endometrial tissues (by about 4- and 9-fold, respectively). Immunofluorescence microscopy revealed co-localization of IPO13 with CD34, CD45, c-kit, telomerase, CD90 and CD146. QPCR revealed significantly increased IPO13 mRNA in endometriosis and endometrial carcinoma versus secretory phase endometrium (by about 2- and 10-fold, respectively). Western blot analysis showed that IPO13 protein is enhanced in endometriosis and endometrial carcinoma versus secretory phase endometrium (p<0.05)., Conclusions: These results demonstrate an increased expression of IPO13 in endometriosis and endometrial carcinoma, which could be involved in the pathogenesis of endometriosis and endometrial carcinoma; IPO13 can serve as an endometrial progenitor/stem cell marker.

A green, one-step method for synthesis of graphene-Au nanoparticles (graphene-AuNPs) was introduced in this article, using an environmentally benign hexamethylenetetramine (HMTA) as reducing and stabilizing agent. HMTA slowly was hydrolyzed to generate aldehyde ammonia to reduce graphene oxides (GO) and hydrogen tetrachloroaurate (Au precursor). The structure and composition of the graphene-AuNPs nanocomposites were studied by means of ultraviolet visible (UV) absorption spectra, X-ray photoelectron spectroscopy (XPS) and Transmission electron microscopy (TEM). The AuNPs are well-dispersed on graphene nanosheets in narrow size range. The nanocomposites have excellent electrocatalytical properties for catalytic reduction of O(2) and H(2)O(2)., (Copyright © 2012. Published by Elsevier B.V.)

Objective: To synthesize 3, 5, 4' -trimethoxystilbene (TMS) by methylation of resveratrol (Res), a natural compound extracted from polygonum cuspidatum, to identify the chemical structure of TMS, to test its pharmacokinetics, and to determine the effects of TMS on the growth inhibition and apoptosis in pulmonary artery smooth muscle cells (PASMCs)., Methods: The chemical structure of TMS was analyzed by UV- and IR- absorption spectrometry, (1)H-NMR and (13)C-NMR spectroscopy and mass spectrometry. We measured the bioavailability, the characteristics of intestinal absorption, and the distribution of TMS in body and excretions of SD rats after oral administration of TMS. The acute toxicity of TMS in mice was tested. PASMCs were prepared from pulmonary artery of SD rats. The PASMCs were divided into 8 groups. Group of A (control) was cultured without TNF-α, TMS, or Res. Group of B (TNF-α) was cultured with 100 pg/mL TNF-α. Groups of C-E (low-high concentrations of TMS) were cultured with 100 pg/mL TNF-α and 5, 10, 20 μmol/L TMS, respectively. Groups of F-H (low-high concentrations of Res) were cultured with 100 pg/mL TNF-α and 50, 100, 200 μmol/L Res, respectively. The proliferation of PASMCs after treatment was determined by MTT assay. The apoptosis of PASMCs after treatment was determined by flow cytometry., Results: The UV absorption map of TMS showed λmax(MeOH) at 318, 306.2, and 217.8 nm. Analysis of infrared spectrum of TMS showed IRvKBr max /cm at 2999, 2935, 2836, 1591, 1511 and 1456/cm. The (1)H-NMR map showed that the synthetic product contained three hydroxy groups, while (13)C-NMR map showed 17 carbon signals and some symmetrical structural fragments. Electospray ionization mass spectrometry of the productshowed m/z peaks corresponded to 271[M+H](+), 256[M+H-CH(3)](+) and 241[256-CH(3)](+); the implied relative molecular weight is 270 and the implied molecular formula is C17H18O3. These data confirm the product is 3,5,4' - trimethoxystilbene. The absolute bioavailability of TMS was 45.4%. TMS was well absorped in the upper small intestine; it was excreted in stool and bile and distributed into several tissues. The maximal tolerance dose (MTD) of TMS was 5.85 g/kg. MTT assay showed TMS inhibited the proliferation of PASMCs in a dose-dependent manner. The extent of growth inhibition in A-H groups were (4.07±2.12)%, (6.54±4.78)%, (9.35±4.26)%, (16.75±5.34)%, (23.74±7.07)%, (6.78±5.58) %, (8.81±5.16) %, and (17.81±6.03) %, respectively. Flow cytometry showed the extent of apoptosis in PASMCs (after being treated with TMS for 24 h) was significantly higher than that in PASMCs treated only with TNF-α. The apoptosis rates of A-H groups were (2.63±0.74)%, (3.54±0.81)%, (5.77±4.62)%, (11.68±5.35)%, (18.79±4.15)%, (4.11±3.59)%, (6.33±4.8) %, and (12.47±5.06)%, respectively., Conclusion: We have confirmed our synthetic product as 3,5,4'-trimethoxystilbene (TMS), with the molecular formula of C17H18O3 and appropriate molecular weight and absorbption and NMR spectra. The bioavailability of TMS was to 45%. It strongly inhibits the proliferation of PASMCs in a dose-dependent manner and induces apoptosis of PASMCs.

Esophageal cancer is the eighth most common cancer and causes the sixth highest cancer-related mortality worldwide. The 5-year survival of patients suffering from esophageal cancer in either advanced stage or metastasis is less than 20%. MicroRNAs are small, well conserved, non-coding RNA molecules that either repress translation or promote mRNA degradation based on the degree of complementary between miRNAs and mRNAs. Based on biogenesis and function of microRNAs, specific microRNA profiles, either from cancerous tissues or serum, were able to serve as diagnostic and prognostic biomarkers of esophageal cancer and predicted the effectiveness of surgery and chemoradiotherapy. MicroRNAs could also influence the biological behaviors of esophageal cancer cells, such as cellular proliferation, apoptosis, invasion and metastasis. MicroRNAs were also associated with multi-drug resistance of esophageal cancer. Further studies on the roles of microRNAs in esophageal cancer would provide a strategy to prevent and treat esophageal cancer, and reverse multi-drug resistance of esophageal cancer.

Objective: To explore the effect of unidirectional valved patch (UVP) for congenital heart disease (CHD) with severe pulmonary hypertension (PH)., Methods: We retrospectively analyzed the treatment of 37 CHD patients with severe PH by UVP in the operation, and summarized its short-term to mid-term effect to find an optimum therapeutic regimen., Results: Before the operation, the ECG showed that the mean pulmonary artery pressure (MPAP) ranged 65-72 mmHg, and the cardiac catheterization showed the pulmonary artery pressure ranged 80-120 mmHg, P(P)/P(A) ranged 0.8-1.05,PVR ranged 8.5-19.2 (under oxygen inhalation 6.8-14.6) wood unit.After the operation, P(P)/P(A) ranged 0.4-0.72 on weaning-off CPB. Postoperative ECG showed the MPAP ranged 32-48 mmHg. No pulmonary hypertension crisis occurred and no patient died. Mechanical ventilation time ranged from 32 h to 8 d and the SaO₂ ranged 93%-96% at rest after the extubation.The right-to-left shunt situations by ECG were as follows:22 cases had shunt 5 d after the operation, 11 cases had shunt 1 month after the operation,4 cases 3 months after the operation, and none 1 year after the operation but one patient lost follow-up.However,there were no long-term follow-up data: 12 patients had a 1-year follow-up, 5 patients had a 3-year follow-up, and most patients had just 3-month follow-up., Conclusion: UVP can decrease the operative risk in CHD with severe PH at perioperative period. The short-term to mid-term effect is satisfactory, while long-term effect remains uncertain.

To investigate whether alterations in histone modifications occur in esophageal squamous cell carcinoma (ESCC), we measured histone H3/ H4 acetylation and H3K4/H3K27 methylation levels, as well as the expression of chromatin modifier genes in tumor and healthy esophageal tissue from ESCC patients. We found evidence of global H3 and H4 hypoacetylation, H3K4 and H3K27 hypermethylation in ESCC tissue. Both H3 hypoacetylation and H3K27 hypermethylation correlated with the severity and histological differentiation of the tumor, and H3K4 hypermethylation also correlated with tumor differentiation. Our results suggest that aberrant histone modifications may play an important role in the development and progression of ESCC.

Background: To investigate the expression levels of importin13 (IPO13), c-kit, CD146, telomerase, caspase-3, bcl-2 and bax in endometrial polyps (EPs)., Material/methods: We detected the mRNA expression levels of IPO13, c-kit, bcl-2 and bax in endometrial polyps (EPs) using real-time PCR. We detected the protein expression levels of IPO13, telomerase, CD146, caspase-3, bcl-2 and bax in EPs using S-P (Streptavidin-Peroxidase) immunohistochemistry. Western blotting was performed to determine the levels of importin13 and bcl-2 proteins in EPs., Results: The expression levels of IPO13, c-kit, telomerase, caspase3, and bax were lower in the EP tissue compared to normal endometrial tissue during the proliferation and secretion phases of the menstrual cycle (p<0.05). The expression of CD146 was decreased in the EP tissue compared to the normal endometrial tissue during the proliferation phase of the menstrual cycle (p<0.05). The expression of bcl-2 was increased in the EP tissue compared to the normal endometrial tissue during the proliferation and secretion phases of the menstrual cycle (p<0.05)., Conclusions: The expression levels of IPO13, c-kit, telomerase, caspase3, and bax were decreased; however, the expression of bcl-2 was increased in the EP tissue compared to the normal endometrial tissue. These findings suggest that the development of EPs is associated with the deregulated activities of the endometrial stem/progenitor cells and the decreased apoptosis of endometrial cells, with the latter being the major factor involved in the development of EPs.

Reactive astrogliosis impedes axonal regeneration after injuries to the mammalian central nervous system (CNS). Here we report that glial cell line-derived neurotrophic factor (GDNF), combined with transplanted Schwann cells (SCs), effectively reversed the inhibitory properties of astrocytes at graft-host interfaces allowing robust axonal regeneration, concomitant with vigorous migration of host astrocytes into SC-seeded semi-permeable guidance channels implanted into a right-sided spinal cord hemisection at the 10th thoracic (T10) level. Within the graft, migrated host astrocytes were in close association with regenerated axons. Astrocyte processes extended parallel to the axons, implying that the migrated astrocytes were not inhibitory and might have promoted directional growth of regenerated axons. In vitro, GDNF induced migration of SCs and astrocytes toward each other in an astrocyte-SC confrontation assay. GDNF also enhanced migration of astrocytes on a SC monolayer in an inverted coverslip migration assay, suggesting that this effect is mediated by direct cell-cell contact between the two cell types. Morphologically, GDNF administration reduced astrocyte hypertrophy and induced elongated process extension of these cells, similar to what was observed in vivo. Notably, GDNF treatment significantly reduced production of glial fibrillary acidic protein (GFAP) and chondroitin sulfate proteoglycans (CSPGs), two hallmarks of astrogliosis, in both the in vivo and in vitro models. Thus, our study demonstrates a novel role of GDNF in modifying spinal cord injury (SCI)-induced astrogliosis resulting in robust axonal regeneration in adult rats., (Copyright © 2011. Published by Elsevier Inc.)

Aims: Here we aimed to firstly investigate the role of miR-27a in proliferation and multidrug resistance of gastric cancer cells., Methods: The role of miR-27a in gastric cancer cells was detected using MTT assay, soft agar assay, flow cytometry assay, nude mice assay, real-time PCR, western blot and reporter gene assay, etc., Results: Down-regulation of miR-27a could inhibit porliferation of gastric cancer cells in vitro and in vivo. Down-regulation of miR-27a could also confer sensitivity of drugs on gastric cancer cells, and might increase accumulation and decrease releasing amount of adriamycin in gastric cancer cells. Down-regulation of miR-27a could significantly decrease the expression of P-glycoprotein and the transcriptional activity of cyclin D1, and up-regulate the expression of p21., Conclusions: MiR-27a might play important roles in porliferation and drug resistance of gastric cancer. MiR-27a might be considered as a useful target for cancer therapy.

Objective: To summarize the characteristics of reoperative valve surgery after previous open-heart valve surgery., Methods: From 1996 to 2010, 155 patients who underwent reoperative valve surgery, either valve replacement or tricuspid annuloplasty or the repair of perivalvular leakage were included in the study. The reoperative interval was 1-266 (94.82 ± 85.37) months. All surgeries were carried out with extracorporeal circulation under moderated hypothermia. The cardioplegic solution in cold crystal or blood was used if heart beating was stopped during the surgery., Results: The total in-hospital mortality was 5.81%, while it was 2.75% from 2005 to 2010. The end-diastolic dimension, size of atrium and ventricles were reduced after the reoperation. Ventricular arrhythmia and low cardiac output were the most frequent complications., Conclusion: The success rate of reoperative valve surgery can be improved by the distinctive therapeutic strategies based on the clinical characteristics and therapy principles obtained from practice experiences.

Background: It was unclear whether chronic endometrial ischemia contributed to the pathogenesis of thin endometrium and was associated with decreased endometrial stem/progenitor cell. Thus, we explored the role of chronic endometrial ischemia in the pathogenesis of thin endometrium and its effect on endometrial stem/progenitor cells apoptosis., Material/methods: In vitro, endometrial side population (ESP) cell apoptosis models were built, and apoptosis was quantified by fluorescence-activated cell sorter (FACS) analysis, pou5f1, and c-kit mRNA was detected by qPCR. In vivo, a rat model of chronic endometrial ischemia was induced by performing bilateral uterine artery ligation. TERT and caspase3 were detected by immunohistochemistry. Pou5f1and c-kit mRNA was examined by qPCR. C-kit, caspase3 and telomerase were detected by Western blot., Results: In the in vitro endometrial SP (ESP) cells apoptosis model, we found that the apoptotic rate was gradually increased with time, prolonging the expression of TERT, and c-kit mRNA was gradually decreased. In the in vivo endometrial SP (ESP) cells apoptosis model, we found that endometrial thickness, luminal epithelium thickness, gland epithelium thickness and the number of glands in the experiment group were significantly decreased compared with those in the control group (P<0.05). The expression levels of c-kit, pou5f1 and telomerase was significantly lower in the experimental group than those in the control group (P<0.05). The expression level of caspase3 was significantly higher in the experimental group compared with that in the control group (P<0.05)., Conclusions: The present work shows that chronic ischemia and chronic endometrial ischemia-associated stem/progenitor cells apoptosis may be responsible for the pathogenesis of thin endometrium.

The innate immune system, mediated via toll-like receptors (TLRs), represents the first line of defensive mechanisms that protects hosts from invading microbial pathogens. TLRs are a family of pattern recognition receptors (PRRs), and are pathologically activated by a set of pathogen-associated microbial patterns (PAMPs) and damage-associated molecular patterns (DAMPs). TLRs deliver signals via a specific intracellular signaling pathway involving distinctive adaptor proteins and protein kinases, and ultimately initiate transcriptional factors resulting in inflammatory responses. TLR4 is a paramount type of TLRs, located in the heart, and plays an important role in mediating myocardial ischemic reperfusion (I/R) injury. Loss-of-function experiments and animal models using genetic techniques have found that the MyD88-independent and the MyD88-dependent pathways together participate in the pathological process of myocardial I/R injury. Some other distinctive signaling pathways, such as the PI3K/AKt and AMPK/ERK pathways, interacting with the TLR4 signaling pathway, were also found to be causes of myocardial I/R injury. These different pathways activate a series of downstream transcriptional factors, produced a great quantity of inflammatory cytokines, such as IL, TNF, and initiate inflammatory response. This results in cardiac injury and dysfunction, such as myocardial stunning, no reflow phenomenon, reperfusion arrhythmias and lethal reperfusion injury, and other related complication such as ventricular remodeling. In the future, blockades aimed at blocking the signaling pathway could benefit developments in pharmacology.,

Objective: To summarize the resection of local advanced upper lung cancer and radical bilateral mediastinal lymph node dissection through a median sternotomy., Methods: A total of 31 patients with local advanced upper lung cancer underwent lobectomy and radical complete dissection of bilateral superior mediastinal lymph node through a median sternotomy (the sternotomy group). The sternotomy group consisted of 8 females and 23 males, from 35 to 75 years old (average 57 years). Five patients underwent superior vena caval replacement or partial excision, 21 underwent upper sleeve lobectomy, and 6 patients combined with right pulmonary artery sleeve angioplasty or partial resection and reconstruction. Compared with the 30 patients who were operated through posterolateral incision, the surgery time, complications, and prognosis during the same period (the posterolateral incision group) were recorded., Results: There was no perioperative death. The average operation time in the sternotomy group was (170±30)min, while that in the posterolateral incision group was (140±30) min(P>0.05). Postoperative complications comprised atelectasis, cardiac arrhythmia, and pneumonia. In the sternotomy group it was 6.5%(2/31), 16.1%(5/31), and 6.5% (2/31),and that in the posterolateral incision group 3.3%(1/30), 20%(6/30), 10.0%(3/30),respectively. Postoperative pathological findings demonstrated the rate for pN3 disease in the sternotomy group was 29%(9/31), 2 patients died of brain and liver metastasis respectively 10 or 11 months after the operation. The 3 year survival rate of 9 patients with pN3 diagnosed as cN2 preoperatively was 33.3%(3/9). The total survival rate of 1,3 years in the sternotomy group was 90.3%(28/31) and 41.9%(13/31), in the posterolateral incision group 86.6%(26/30) and 40.0%(12/30),respectively(P>0.05)., Conclusion: Median sternotomy helps to resect local advanced upper lung cancer completely and to dissect bilateral mediastinal lymph node, and it can also provide more complete postoperative lymph node staging with no significant increase in complications.

Objective: To observe the clinical results of laminated anastomosis using absorbable suture in cervical esophagogastrostomy, and to reduce the incidence of cervical esophagogastric anastomotic stricture., Methods: A retrospective analysis was carried out on 210 patients who underwent cervical esophagogastrostomy after subtotal esophagectomy from January 2008 to June 2010. Among them, 96 cases were treated with traditional full layer interrupted varus suture (varus group) and the remaining 114 cases were treated with seromuscular layer and mucosal layer laminated anastomosis with absorbable suture (laminated group). Esophageal angiography was performed in 1 week, 1 month, and 3 months after the operation. The diameter of anastomatic stoma was measured on the anteroposterior and lateral angiography image respectively. The area of anastomatic stoma was calculated. The degree of stenosis was assessed according to the patients' dysphagia symptom., Results: There was no operative deaths, no serious pulmonary complications and chylothorax, no sever esophageal reflux in all patients. The ratio of cervical esophagogastric anastomotic leakage was 2.1% (2/96) in the varus group. No anastomotic leakage in the laminated group. Compared with the varus group, the area of the anastomatic stoma in the laminated group was significantly increased in all measured time points (P<0.01). The incidence of obstruction in the laminated group was decreased significantly (P<0.01) in 1 month or in 3 months after operation compared with the varus group., Conclusion: Application of the laminated anastomosis with absorbable suture in cervical esophagogastrostomy can significantly reduce the incidence of anastomotic stenosis.

The transplantation of Schwann cells (SCs) could successfully promote axonal regeneration. This is likely to attribute to the adhesion molecules expression and growth factors secretion of SCs. But which factor(s) play a key role has not been precisely studied. In this study, an outgrowth assay using dorsal root ganglia (DRG) neuron-SC co-culture system in vitro was performed. Co-culture of SCs or application of SC-conditioned medium (CM) substantially and significantly increased DRG neurite outgrowth. Further, nerve growth factor and NGF receptor (TrkA) mRNA were highly expressed in Schwann cells and DRG neuron, respectively. The high concentration of NGF protein was detected in SC-CM. When K-252a, a specific inhibitor of NGF receptor was added, DRG neurite outgrowth was significantly decreased in a concentration-dependent manner. These data strongly suggest that SCs play important roles in neurite outgrowth of DRG neurons by secreted NGF.

Objectives/hypothesis: Aortoesophageal fistula is a rare but life-threatening complication of foreign body ingestion. In spite of several strategies for treatment, there is little consensus regarding the optimal management of the entity. In this article, we present our experience in the diagnosis and treatment of patients with aortoesophageal fistula., Study Design: Retrospective study., Methods: A total of 3,209 admissions due to esophageal foreign body impaction were recorded in Second Xiangya Hospital between 1963 and 2010. Of these, 32 cases were complicated with aortoesophageal fistula. In these 32 patients, 19 were treated by open surgery and 13 were managed with nonsurgical measures. We compared different treatments of the patients and their clinical outcomes., Results: Foreign body and aortoesophageal fistula were found in all of these 32 cases in imaging examination and/or surgery. Three patients were completely cured and 16 patients died of fatal hemorrhage from the group of patients with surgical management. All 13 nonsurgically treated cases died., Conclusions: Early diagnosis and an aggressive surgical treatment without delay is the only form of effective therapy for the condition of aortoesophageal fistula. Imaging examination is helpful in diagnosis. In particular, three-dimensional computerized tomography is a safe, simple, and noninvasive examination method that has high sensitivity and specificity for the early diagnosis of aortoesophageal fistula., (Copyright © 2011 The American Laryngological, Rhinological, and Otological Society, Inc.)

We describe a new procedure of aortic valvuloplasty for aortic valve prolapse in ventricular septal defect with aortic insufficiency syndrome. This technique allows an anatomic and functional aortic valve reconstruction that prevents late failure of aortic valve repair and reoperation. Midterm results demonstrate the feasibility and durability of this new procedure., (Copyright © 2011 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.)

Regeneration deficiency is one of the main obstacles limiting the effectiveness of tissue-engineered scaffolds. To develop scaffolds that are capable of accelerating regeneration, we created a heparin/chitosan nanoparticle-immobilized decellularized bovine jugular vein scaffold to increase the loading capacity and allow for controlled release of vascular endothelial growth factor (VEGF). The vascularization of the scaffold was evaluated in vitro and in vivo. The functional nanoparticles were prepared by physical self-assembly with a diameter of 67-132 nm, positive charge, and a zeta potential of ∼30 mV and then the nanoparticles were successfully immobilized to the nanofibers of scaffolds by ethylcarbodiimide hydrochloride/hydroxysulfosuccinimide modification. The scaffolds immobilized with heparin/chitosan nanoparticles exhibited highly effective localization and sustained release of VEGF for several weeks in vitro. This modified scaffold significantly stimulated endothelial cells' proliferation in vitro. Importantly, utilization of heparin/chitosan nanoparticles to localize VEGF significantly increased fibroblast infiltration, extracellular matrix production, and accelerated vascularization in mouse subcutaneous implantation model in vivo. This study provided a novel and promising system for accelerated regeneration of tissue-engineering scaffolds.

Objective: To summarize the successful experience of 45 patients with minimally invasive mitral valve surgery via right minithoracotomy., Methods: Forty-five patients with mitral valve disease were enrolled. A main surgical wound (4-6 cm)was made over the lateral border of the right breast. Cardiopulmonary bypass was established via femoral cannulation. The Chitwood clamp was inserted thlution the 2nd or 3rd intercostal space. After the cross-clamp was placed, cold blood cardioplegic solution was delivered via right minithoracotomy. The procedures of mitral valve replacement were performed via left atrialotomy., Results: All the operations were successful. There was no death. The duration of operation and extracorporeal circulation was 80-291(139±35) min, and 25-110(49±21) min, respectively. The cross-clamped time was 18-77 (33±12) min, the ventilation time was 4.5-45(14.8±10) h, the total chest tube drainage was 50-1 050(262±110) mL, and the postoperative hospital stay was 5-14(7.5±1.8) d., Conclusion: Minimally invasive mitral valve surgery via right minithoracotomy is feasible, safe, and less invasive with rapid recovery,and has superior cosmetic results for patients.

Here we firstly investigated the role of miR-138 in multidrug resistance of leukemia cells. miR-138 was found up-regulated in the vincristine-induced multidrug resistance (MDR) leukemia cell line HL-60/VCR as compared with HL-60 cells. Up-regulation of miR-138 could reverse resistance of both P-glycoprotein-related and P-glycoprotein-non-related drugs on HL-60/VCR cells, and promote adriamycin-induced apoptosis, accompanied by increased accumulation and decreased releasing amount of adriamycin. miR-138 could significantly down-regulate the expression of P-glycoprotein, Bcl-2, and the transcription of the multidrug resistance gene 1. Further study of the biological functions of miR-138 might be helpful for developing possible strategies to treat leukemia., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

Purpose: Transforming growth factor beta receptors II gene (TGFBR2) mutations associated with Marfan syndrome and Marfan-associated disorders have been investigated. However, such studies are limited in China. To obtain more information about TGFBR2 mutations, we analyzed 6 unrelated Chinese patients with Marfan-associated disorders and without ocular manifestation., Methods: The genomic DNA from blood leukocytes of these 6 patients and their relatives was isolated, and the entire coding region of TGFBR2 was amplified using PCR. We determined the sequence of TGFBR2 with the ABI 3100 Genetic Analyzer., Results: Three mutations were identified in TGFBR2. Two mutations were associated with Loeys-Dietz syndrome (LDS), which were distributed as following: one missense mutation R528C (caused by a 1582C > T substitution) and one polymorphism T315M (a rare SNP). The third mutation was a novel silent mutation associated with MFS2, which was K291K caused by an 873 C > T substitution., Conclusions: The TGFBR2 gene missense mutations are possibly causative mutations of Loeys-Dietz syndrome. This result suggests an increase in the mutation spectrum of Marfan-related disorders in China and possibly world-wide.

The extracellular matrix (ECM) is a component of neural cell niches and regulates multiple functions of diverse cell types. To date, limited information is available concerning its biological effects on the growth properties of oligodendrocyte progenitor cells (OPCs). In the present study, we examined effects of several ECM components, i.e., fibronectin, laminin, and Matrigel, on the survival, proliferation, migration, process extension, and purity of OPCs isolated from embryonic day 15 rat spinal cords. All three ECM components enhanced these biological properties of the OPCs compared with a non-ECM substrate, poly-D-lysine. However, the extents of their effects were somewhat different. Among these ECMs, fibronectin showed the strongest effect on almost all aspects of the growth properties of OPCs, implying that this molecule is a better substrate for the growth of OPCs in vitro. Because of its survival- and growth-promoting effects on OPCs, fibronectin may be considered as a candidate substrate for enhancing OPC-mediated repair under conditions when exogenous delivery or endogenous stimulation of OPCs is applied., (2009 Wiley-Liss, Inc.)

Objective: To study the inflammation response and the biocompatibility of valved bovine jugular vein conduit (BJVC) and valved bovine jugular vein patch (VBJV-P) in treating complex congenital heart disease (CHD)., Methods: From December 2007 to March 2008, 16 patients with complex CHD were treated. Of 16 patients, 6 underwent conjunction right ventricular to pulmonary artery with BJVC and broaden right ventricular outflow tract (RVOT) with VBJV-P (BJVC group), and 10 underwent broaden RVOT with self pericardial patch (control group). In BJVC group, there were 3 males and 3 females, aging (5.6 +/- 3.6) years, and including 1 case of type I truncus arteriosus, 1 case of type I truncus arteriosus with ventricular septal defect and patent foramen ovale, 1 case of congenital pulmonary atresia with ventricular septal defect and patent arterial duct, and 3 cases of Fallot's tetrad. In control group, there were 5 males and 5 females, aging (4.3 +/- 3.1) years, all being Fallot's tetrad. The periphery vein blood of the two groups was collected during operation and after operation, and the levels of cytokine were detected with ELISA method. Meanwhile the clinical data of the two groups were collected., Results: There were no significant differences at levels of TNF-alpha and IL-6 between BJVC group and control group 1 week after operation (P > 0.05), and there was significant difference at level of IL-10 [(25.7 +/- 5.0) pg/mL vs (19.5 +/- 4.7) pg/mL, P < 0.05]. There were no significant differences at levels of IL-6 and IL-10 within groups botvin control group and in BJVC group (P > 0.05) between 1 week after operation and the anesthesia inducing period. And there was significant difference at level of TNF-alpha in BJVC group [(77.0 +/- 1.6) pg/mL vs (82.9 +/- 1.3) pg/mL, P < 0.05] and in control group [(78.6 +/- 3.4) pg/mL vs (83.1 +/- 1.9) pg/mL, P < 0.05] between 1 week after operation and the anesthesia inducing period. There were no statistically significant differences (P > 0.05) in leukocyte count and body temperature between BJVC group and control group. The X-ray films showed no abnormality in BJVC group and control group before operation and after operation. No hepatic and renal dysfunction occurred in control group; and 2 patients had hepatic dysfunction, which may be caused by antibiotics., Conclusion: BJVC has a good biocompatibility in treating complexity CHD.

The present study was conducted to investigate whether Ginkgo biloba extract (EGb) 761 could protect spinal cord neurons from H(2)O(2)-induced toxicity. In primary spinal cord neurons isolated from embryonic day 14 rats, H(2)O(2) administration resulted in a significant decrease in the survival of spinal cord neurons. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and Hoechst 33342 nuclear staining showed that these cells die by apoptosis. Such neuronal death, however, was significantly reversed by EGb761 in a dose-dependent manner. Moreover, a marked increase in intracellular free radical generation was found after the H(2)O(2) administration which could be reversed almost completely by EGb761, indicating that inhibition of free radical generation is an important mechanism of the anti-apoptosis action of EGb761. Finally, treatment of cells with H(2)O(2) for 12 h reduced the expression of Bcl-2, an anti-apoptotic gene, by 70% but showed no effect on the level of Bax, a pro-apoptotic gene. EGb76 treatment, however, significantly reversed H(2)O(2)-induced reduction of Bcl-2 expression and inhibited Bax expression by 2.3-fold. Thus, our study provided evidence showing that the protective effect of EGb761 on spinal cord neuronal apoptosis after oxidative stress is mediated, at least in part, by its anti-oxidative action and regulation of apoptosis-related genes Bcl-2 and Bax.

Aberrant DNA methylation plays a critical role in the development and progression of many types of cancer. To investigate whether DNA methylation is abnormal in thymic epithelial tumors (TETs), we analyzed global methylation levels and the methylation status of 9 tumor suppressor gene (TSG) promoters in 65 TET samples. We found evidence of TSG promoter hypermethylation and decreased TSG expression in severe TETs. Furthermore, relative to early-stage TETs, global DNA methylation levels were reduced and DNA methyltransferase expression was increased in advanced-stage TETs. Our results suggest that aberrant DNA methylation is associated with TET development.

Grafting oligodendrocyte precursor cells (OPCs) has been used as a strategy to repair demyelination of the central nervous system (CNS). Whether OPCs can promote CNS axonal regeneration remains to be tested. If so, they should be permissive to axonal growth and may express less inhibitory molecules on their surface. Here we examined the expression of two oligodendrocyte-associated myelin inhibitors Nogo-A and myelin-associated glycoprotein (MAG) during oligodendrogliogenesis and tested their abilities to promote neurite outgrowth in vitro. Whereas the intracellular domain of Nogo-A was consistently expressed throughout oligodendrocyte differentiation, MAG was expressed only at later stages. Furthermore, the membrane-associated extracellular domain of Nogo-A was not expressed in OPCs but expressed in mature oligodendrocytes. In a dorsal root ganglion (DRG) and OPC/oligodendrocyte co-culture model, significantly greater DRG neurite outgrowth onto OPC monolayer than mature oligodendrocyte was found (1042+/-123 vs. 717+/-342 micrometer; p=0.011). Moreover, DRG neurites elongated as fasciculated fiber tracts and contacted directly on OPCs (133+/-37 cells/fascicle). In contrast, few, if any, direct contacts were found between DRG neurites and mature oligodendrocytes (5+/-3 cells/fascicle, p<0.001). In fact, acellular spaces were found between neurites and surrounding mature oligodendrocytes in contrast to the lack of such spaces in OPC/DRG coculture (51.1+/-16.5 vs. 2.4+/-3.9 micrometer; p<0.001). Thus, OPCs expressing neither extracellular domain of Nogo-A nor MAG are significantly more permissive than mature oligodendrocytes expressing both. Grafting OPCs may thus represent a feasible strategy to foster CNS axonal regeneration.

Objective: To evaluate the effect of different control levels of glucose on the serum lactic acid during operation, and to investigate the relation between glucose and lactic acid to find a new way of myocardial protection., Methods: Volunteers were divided into an experiment group(n=38) and a control group(n=33) by random sampling and double blind method. The experiment group received intensive insulin therapy and the control group received traditional therapy. The arterial blood gas samples of all the patients at different time points after the operation were harvested in the intensive care unit for blood gas analysis. The related data were collected and analyzed., Results: The serum glucose level in the 2 groups decreased firstly, then increased, and recovered finally. The serum lactic acid level in the 2 groups increased firstly, decreased later, then reincreased, and recovered finally. The highest level of the serum lactic acid was found 2 hours after the operation. There were significant differences in serum glucose and lactic acid levels at 2, 12, and 24 h after the operation in the two groups (P<0.01). The other data were not significant (P>0.05)., Conclusion: The variation of serum glucose and lactic acid level at 2, 12, 24 h after the valve replacement is consistent and significant. The serum lactic acid in the serum may be decreased by controlling the blood glucose, which provides experiment basis for myocardial protection.

Objective: To explore the effect of vascular smooth muscle alpha-actin(VSMA-alpha) on cardiac allograft vasculopathy and fibrosis., Methods: After heterotopic cardiac transplantation model was established, the rats were divided into an acute rejection group, a chronic rejection group, an isograft group, and a normal group. Van Gieson staining and Massonos trichrome staining were used to observe the cardiac allograft vasculopathy and fibrosis respectively, and immunohistochemical stainings were used to examine VSM-alpha expression., Results: The fibrosis score of the normal group, the isograft group, the acute rejection group and the chronic rejection group were 0.00+/-0.00, 0.63+/-0.52, 1.75+/-0.71, and 2.75+/-0.46, respectively. The fibrosis score of the chronic rejection group increased significantly as compared with that of the normal group and the isograft group (both P<0.01), the indexes of coronary arteries stenosis were 0.08+/-0.02, 0.09+/-0.04, 0.12+/-0.05, and 62.86+/-17.18, respectively. The stenosis index in the chronic rejection group increased significantly as compared with that of all the other groups (P<0.01).VSMA-alpha was abundant in the allograft myocardium in the chronic rejection group., Conclusion: Reactivation of fetal structural genes of vascular smooth muscle alpha-actin and its ectopic expression is associated with the cardiac allograft vasculopathy and fibrosis.

Objective: To analyze the clinic and pathologic data of thymic epithelial tumor (TET) and to explore its prognostic factors., Methods: From June 1997 to September 2007, 137 patients with TET were surgically treated in our hospital. The data included age, gender, symptoms, histological type, stage and grade, pathological findings, and operation reports. The patients were followed up by telephones and mails. The patients were divided into Masaoka I/II group and III/IV group, and WHO A/AB/B1 group and B2/B3/C group. Kaplan-Meier method, log-rank test, and COX regression model were used to analyze the prognostic factors for TET., Results: Among the 137 patients, 124 (90.5%) received complete resection, 9 (6.6%) incomplete resection, and 4 (2.9%) surgical biopsy. The rate of complete resection was significantly higher in Masaoka stages I/II than that in stages III/IV (P<0.001). The overall 5-year and 10-year survival rate was 71.4å and 50.1å, respectively. Patients in stage I/II had better long-term survival than those in stage III/IV (P<0.001). According to WHO histological classification, the 5-year and 10-year survival rate in patients with Type A/AB/B1 TET was significantly higher than that in patients with Type B2/B3/C TET (P<0.001). The 5-year and 10-year survival rate in patients with complete resection was significantly higher than that in patients with incomplete resection and biopsy (P<0.001).Cox regression analysis showed that the prognosis of patients with TET was related to Masaoka stage, WHO histological classification, extent of resection, and age at operation., Conclusion: Masaoka stage, WHO histological classification, extent of resection, and age at operation are important prognostic factors in patients with TET.

Objective: To construct the eukaryotic expression vector hypoxia-inducible factor 1alpha-pcDNA(3.1) and to investigate its transfective efficiency into mesenchymal stem cells (MSCs) in vitro and the expression of HIF-1alpha gene in MSCs., Methods: mRNA of Wistar Rats' myocardial cells was extracted, and cDNA was synthesized with Reverse Transcription Kit, HIF-1alpha was amplified by polymerase chain reaction (PCR), and constructed into pcDNA(3.1). Transfected HIF-1alpha-pcDNA(3.1) into MSCs by liposome mediated method. The expression of HIF-1alpha in the cells was detected by Western Blot Analysis and ELISA., Results: Eukaryotic expression vector HIF-1alpha-pcDNA(3.1) was constructed successfully. Analyzed by flow cytometer, The MSCs' surfaces mark were CD44+, SH3(CD73)+, CD34-, CD45- and the CD44+ cells and SH3(CD73)+ cells were 94.7% and 97.3%, respectively, showing the high purity of the cultured MSCs. After inducing, the cultured MSCs can differentiate into osteoblasts and adipocytes successfully. In HIF-1alpha gene transfected MSCs, the expression of HIF-1alpha mRNA and HIF-1alpha protein were both increased obviously., Conclusion: HIF-1alpha was cloned successfully. HIF-1alpha-pcDNA(3.1) can be transfected into MSCs by liposome-mediated method effectively and which resulting stable expression of HIF-1alpha in transfected MSCs.

In our study we use nordihydroguaiaretic acid (NDGA), the naturally occurring lignan, to investigate whether it plays a role in the prevention and treatment of cancer by epigenetic modifications. The growth inhibitory effect of NDGA on human breast cancer cell lines was determined using the MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay). It substantially inhibited the growth of human breast cancer cell lines SKBR3 and MDA-MB-435 with an estimated IC50 of 31.09+/-1.6 and 38.8+/-2.1 micromol/l respectively, after 4 days incubation with different NDGA concentrations. The in-vivo anticancer activity of NDGA was evaluated by calculating the tumor growth inhibition value. NDGA substantially inhibited the growth of human breast carcinoma cells in both animal and cell-based models. We also found that a single treatment with NDGA reactivates methylation-silenced E-cadherin gene in vitro and in vivo, suggesting an intriguing concept that lignans may act as natural effective epigenetic modifiers in the prevention and treatment of cancer.

Alteration in transforming growth factor-beta (TGF-beta) signaling pathway is one of the main causes of esophageal squamous cell carcinoma (ESCC). The human runt-related transcription factor 3 (RUNX3), an important component of TGF-beta pathway which is located at 1p36, is commonly deleted in a variety of human cancers, including ESCC. Hypermethylation of RUNX3 promoter was frequently found in gastrointestinal cancers, including those of stomach, liver, colon and pancreas. However, RUNX3 promoter methylation status in ESCC has not been studied. The aim of this study was to determine whether promoter methylation of the RUNX3 gene correlates with ESCC tumor progression.Accordingly, we first determined RUNX3 mRNA expression and methylation status of its promoter region in 42 primary tumors with ESCC and Eca-109, an ESCC cell line. Loss of RUNX3 mRNA expression was detected by RT-PCR in 23 out of 42 (54.8%) ESCC specimens and Eca-109 cells. The Promoter hypermethylation was detected by Methylation Specific Polymerase Chain Reaction (MS-PCR) in 27 out of 42 (64.3%) ESCC specimen and Eca-109 cells. Importantly, we found positive correlations, not only between the promoter hypermethylation and tumor clinical pathologic stages (P = 0.003), but also between the loss of RUNX3 mRNA expression and the tumor progression (P = 0.016). Finally, we observed that the loss of RUNX3 mRNA expression is statistically correlated with the promoter hypermethylation in these tumors (P < 0.001). Our results suggest that epigenetic silencing of RUNX3 gene expression by promoter hypermethylation may play an important role in ESCC development.

Neural precursor cells (NPCs) have been experimentally used to repair the damaged nervous system either by exogenous transplantation or by endogenous activation. In post-injury inflammation, an array of cytokines including interleukin-1beta (IL-1beta) are released by host as well as invading immune cells and increased markedly. In the present study, we investigated the effects of IL-1beta on the survival, proliferation, differentiation and migration of NPCs as well as underlying intracellular signaling pathways. NPCs derived from the E16 rat brain were expanded in neurospheres that were found to express IL-1beta, IL-1RI and IL-1RII, but not IL-1alpha and IL-1ra. IL-1beta inhibited the proliferation of NPCs in a dose-dependent manner, an effect that can be reversed by IL-1ra, an antagonist for IL-1 receptor. This inhibitory effect of IL-1beta on NPCs proliferation resulted in part from its effect on increased apoptosis of NPCs. Moreover, IL-1ra did not affect NPCs lineage fate but rather inhibited GFAP expression in differentiated astrocytes. We also found that IL-1ra had no effect on the transmigration of NPCs in vitro. Finally, we showed that the effect of IL-1beta on NPCs proliferation and differentiation appeared to be mediated by SAPK/JNK, but not ERK, P38MAPK nor NF-kappaB pathways. These findings collectively suggest that the inflammatory environment following CNS injuries may influence the ability of NPCs to repair the damage.

Objective: To establish the transfection method of vascular endothelial growth factor (VEGF) gene into mesenchymal stem cells (MSCs), to investigate the effect of this gene-transfected MSCs for heart function restoration and angiogenesis after myocardial infarction, and to compare the therapeutic differences among cell therapy, gene therapy, and combined therapy., Methods: Ischemic heart models were constructed in inbred Wistar rats by ligation of the left anterior descending coronary artery. MSCs of Wistar rats were isolated by density gradient centrifugation and purified on the basis of their ability to adhere to plastic, and identified by checking the surface markers and their differentiation capacity, and then followed by transfection of pcDNA(3.1)-hVEGF(165) using the liposome-mediated method. The expression of hVEGF(165) in the transfected cells was detected by Enzyme-Linked Immunosorbent Assay, Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and Western Blot Analysis. The ligated animals were randomly divided into four groups (12 in each) and, after 2 weeks, were injected at the heart infarct zone with hVEGF(165)-transfected MSCs (Combo group), MSCs (Cell group), liposome-hVEGF gene plasmid (Gene group), or medium (Control group). And other six ligated rats (without any injection) were used as Model-assessment group for the baseline heart infarcted size evaluation, and other 12 non-ligated rats (Non-ischemic group) were used as the normal control. Four weeks after the injection, the rats' cardiac function was measured by the Buxco system. Brdu and Troponin-T double labeling and factor VIII were identified by immunohistochemical staining to demonstrate the survival and differentiation of engrafted cells or to evaluate the angiogenesis in the injured heart area; heart infarcted size was calculated by Evan's blue staining. VEGF expression was evaluated by RT-PCR., Results: MSCs can be successfully isolated and cultured by density gradient centrifugation followed by adherence-separation. The cultured MSCs were CD34-, CD45-, CD44+ and SH+. They can differentiate into osteoblasts and adipocytes successfully. The expression of hVEGF(165) in the transfected MSCs was demonstrated with Enzyme-Linked Immunosorbent Assay, RT-PCR and Western Blot Assay. Four weeks after the cells were transplanted, among all groups but the Non-ischemic group, the Combo group had the smallest heart infarcted size and the best heart function. The capillary density of the Combo group was significantly greater than those of both Cell and Control groups. The heart infarcted size, heart function and capillary density of both Cell and Gene groups were similar with each other and smaller, better and greater than those of the Control group, respectively. Brdu and Troponin-T double staining detected a varied increase in the number of survived cardiomyocytes at the heart infarcted area, some of which were double stain positive. RT-PCR showed that the hVEGF(165) gene was expressed in the Combo and Gene groups, and that the former was higher than the latter., Conclusions: Eukaryotic expression vector pcDNA(3.1)-hVEGF(165) can effectively be expressed in MSCs. Transplantation of VEGF gene-transfected MSCs can bring better improvement in myocardial perfusion and in restoration of heart function than either cellular or gene therapy alone.

Objective: To investigate effect of the removal of epithelium and mixed glands from the tracheal allografts on the graft-immunosuppression., Methods: Fresh untreated tracheal allografts, cryopreserved tracheal allografts, and 10 off-epithelium tracheal allografts were obtained from 25 male SD rats. Fresh untreated tracheal allografts (40) were divided into 4 groups and dipped respectively in the solution of protease XIV in 0, 0. 1, 0.3 and 0.5 mg/ml at 4 C for 12 hours. Thirty recipient male SD rats were randomly and equally divided into group A (fresh untreated tracheal allografts), group B (cryopreserved tracheal allografts), and group C (off-epithelium tracheal allografts). The transplanted allografts were implanted into the abdominal cavity of other rats by being embedded in the greater omentum. Twenty-one days after transplantation, the tracheal graft segments were surgically removed, and then were initially fixed in cold 10% neutral buffered formalin solution for hematoxylineosin staining. Histological observation and lymphocyte infiltration were performed on the grafts to evaluate rejection., Results: The 0.3 mg/ml protease XIV could remove the epithelium and mixed glands of the grafts completely, but did no damage to cartilage. The cartilages of each group all survived and were revascularized. The lumens of group A were filled with granulation and necrosis tissue. In contrast, group B was filled with a few granulation tissues and group C was not at all. The number of lymphocyte infiltration in group A, B, and C was 29.16 +/- 2.69/HP, 15.17 +/- 2.19/HP, and 11.56 +/- 0.87/HP respectively. There was significant difference between group A and both group B and group C (P < 0.05), and there was significant difference between group B and group C (P < 0.05). Therefore, the grade of graft-rejection was group A > group B > group C., Conclusion: The 0.3 mg/ml protease XIV can completely remove the epithelium and mixed glands of grafts at 4 C for 12 hours, and it preserves the normal structure of cartilage. The antigenicity of tracheal grafts can be greatly reduced by removing the epithelium and by the cryopreservation. The prior tracheal allograft in the omentum is feasible for the revascularization of the grafts.

Objective: To study the research advance in tracheal allografts undergoing revascularization and reepithelialization., Methods: The recent literature concerned was reviewed. The tracheal allografts are embedded in the omentum, which they were revascularized and reepithelialized by planting in self-epithelia, then the allografts with their omental pedicles were transplanted orthotopically to the cervical or the thoracic portion of the trachea., Results: Compared with the one-stage tracheal allograft approach using the greater omentum, the two-stage approach could increase the successful rate of revascularization and reepithelialization, and made the allografts accord with their physiology., Conclusion: If the approach is successful, it can reduce graft-rejection, prevent graft-collapse and increase graft-viability after tracheal allograft.

Iron and oxygen are essential but potentially toxic constituents of most organisms, and their transport is meticulously regulated both at the cellular and systemic levels. Compartmentalization may be a homeostatic mechanism for isolating these biological reactants in cells. To investigate this hypothesis, we have undertaken a genetic analysis of the interaction between iron and oxygen metabolism in Drosophila. We show that Drosophila iron regulatory protein-1 (IRP1) registers cytosolic iron and oxidative stress through its labile iron sulfur cluster by switching between cytosolic aconitase and RNA-binding functions. IRP1 is strongly activated by silencing and genetic mutation of the cytosolic superoxide dismutase (Sod1), but is unaffected by silencing of mitochondrial Sod2. Conversely, mitochondrial aconitase activity is relatively insensitive to loss of Sod1 function, but drops dramatically if Sod2 activity is impaired. This strongly suggests that the mitochondrial boundary limits the range of superoxide reactivity in vivo. We also find that exposure of adults to paraquat converts cytosolic aconitase to IRP1 but has no affect on mitochondrial aconitase, indicating that paraquat generates superoxide in the cytosol but not in mitochondria. Accordingly, we find that transgene-mediated overexpression of Sod2 neither enhances paraquat resistance in Sod1+ flies nor compensates for lack of SOD1 activity in Sod1-null mutants. We conclude that in vivo, superoxide is confined to the subcellular compartment in which it is formed, and that the mitochondrial and cytosolic SODs provide independent protection to compartment-specific protein iron-sulfur clusters against attack by superoxide generated under oxidative stress within those compartments.

Intraocular injections of ciliary neurotrophic factor (CNTF) or intraocular adenovirus-mediated CNTF gene transfer have been reported to inhibit retinal alterations in inherited retinal degeneration in mice strains. To investigate whether or not CNTF administered by eye drops prevents retinal degeneration in streptozotocin (STZ)-induced diabetic rats, recombinant CNTF was administered to eyes of diabetic rats (n=20) twice daily for 1 month after the onset of diabetes. The b-wave amplitude of electroretinogram in CNTF-administered diabetic rats was significantly larger than that of diabetic rats, and approached that of the controls. Atrophy of the inner plexiform layer, and cavity formation in the pigment epithelium, which were observed in diabetic rats, were prevented in CNTF-administered diabetic rats. These results indicate that CNTF administration by eye drops prevents retinal degeneration in STZ-induced diabetic rats.

Oxidative stress has been widely implicated as an important factor in the aging process. Because mitochondrial respiration is the principal source of reactive oxygen within cells, the mitochondrially localized superoxide dismutase (SOD) 2 is thought to play an important front-line defensive role against aging-related oxidative stress. Although genetic studies with mutants deficient in SOD1, the predominantly cytosolic isoform of SOD, have been instrumental in elucidating the role of reactive oxygen metabolism in aging in Drosophila, the lack of available mutations in the Sod2 gene has hampered an equivalent analysis of the participation of this important antioxidant enzyme in the Drosophila aging model. Here we report that ablation of mitochondrial SOD2 through expression of a GAL4-regulated, inverted-repeat Sod2 RNA-interference transgene in an otherwise normal animal causes increased endogenous oxidative stress, resulting in loss of essential enzymatic components of the mitochondrial respiratory chain and the tricarboxylic acid cycle, enhances sensitivity to applied oxidative stress, and causes early-onset mortality in young adults. In sharp contrast, ablation of SOD2 has no overt effect on the development of larvae and pupae, which may reflect a fundamental transition in oxygen utilization andor reactive oxygen metabolism that occurs during metamorphosis from larval to adult life.

Early pathological and electro-physiological changes of the retina in the streptozotocin (STZ)-diabetic rats were investigated through optical and electron microscopy in two strains and electro-retinography in one strain. In Sprague-Dawley (SD) rats I month after the onset of diabetes, the thickness of the inner plexiform layer (IPL) and photoreceptor segment layer (PSL) was significantly reduced by 9.9% and 18.9%, respectively (P < 0.01, P < 0.05). In Brown-Norway (BN) rats STZ-diabetic for 1 month, the thickness of the IPL was also significantly reduced by 15.7% (P < 0.05). Cytochemical study using peanut agglutinin (PNA), a lectin binding selectively to the cone photoreceptor-associated domains of the inter-photoreceptor matrix, revealed a marked reduction in intensity, number and length of the PNA-binding cone photoreceptors. Electron microscopy showed deepened hollows in the basal infoldings of the retinal pigment epithelium (RPE) of STZ-rats diabetic for 1 month and large concavities into the cytoplasm in STZ-rats diabetic for 6 months. Blood vessels in the retina and choroid were unremarkable. Single-flash electro-retinogram revealed a reduction in the amplitudes of alpha- and beta-waves of electro-retinogram (ERG) of 1 month STZ BN rats (P < 0.05). These findings indicate that the degeneration of rods/cones in the PSL and RPE are the most prominent pathological alteration sites in the early stage of diabetic rats.

Midkine (MK) is a heparin-binding growth factor that occurs as a product of the retinoic acid-inducible gene. Alteration of MK expression in ischemic brain lesions was examined in humans immunohistochemically in nine patients and in two control subjects without neurological disorders. Some neurons were MK-immunopositive, but no evident MK-immunoreactivity was observed in astrocytes in brains of control subjects. In the ischemic lesions, significant elevation of MK-immunoreactivity in the astrocytes and depletion of the reactivity in neurons were seen, especially in the early period, where edema and eosinophilic neurons were prominent. On the other hand, MK-immunoreactivity was not observed in hypertrophic and fibrillary astrocytes in the later period. These findings suggest that the MK in astrocytes play some role in the repair process in the early period of the ischemic brain lesions in humans.