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Bone is a highly calcified tissue with 60 wt% inorganic components. It is made up of mineralized collagen fibrils, where the platelet-like hydroxyapatite nanocrystals deposit within the collagen fibrils in an oriented manner. Inspired by the special structure and biological activity of bone, we realize the intrafibrillar mineralization of collagen films with oriented calcium fluoride nanocrystals in vitro. Amorphous calcium fluoride (ACF) precursors are generated by polyacrylic acid through polymer-induced liquid precursor processes. The precursors are ready to infiltrate and fill the gap zones laterally and then diffuse to occupy the whole space inside the collagen longitudinally. Finally, the fully mineralized collagen fibrils exhibit a single-crystal-like structure after transforming precursors to co-oriented nanocrystals under the influence of arranged collagen molecules. Expanding the collagen mineralization from 1D fibrils to 2D films, the growth of mineralized areas on the films with a reaction-limited behavior is found. The kinetic rate of growth is around 0.2-0.3 μm min-1, which depends on the pH of the solution. Furthermore, the mineralized collagen films exhibit an enhanced ability of cell proliferation over the pure collagen matrices. Understanding the mineralization of artificial collagen-based scaffolds may have broad promising potentials for bone tissue regeneration and repair in the future.

The inflammatory response has been implicated in various cardiac and systemic diseases. Epigallocatechin-3-gallate (EGCG), the major polyphenol extracted from green tea, has various biological and pharmacological properties, such as anti-inflammation, anti-oxidative and anti-tumorigenesis. To some extent, the mechanism of EGCG in the inflammatory response that characterizes myocardial dysfunction is not fully understood. The present study aimed to investigate the inhibiting effect of EGCG on lipopolysaccharide (LPS)-induced inflammation in vitro. Treatment with LPS affected rat H9c2 cardiomyocytes and induced an inflammatory response. However, the LPS-induced effects were attenuated after treatment with EGCG. The present results demonstrated that EGCG treatment repressed several inflammatory mediators, such as vascular endothelial growth factor, chemokine ligand 5, chemokine ligand 2, intercellular adhesion molecule-1, matrix metalloproteinase-2, tumor necrosis factor-α and nitric oxide (induced by LPS), and the repressing effect of EGCG on inflammatory response was dose-dependent in the range of 6.25-100 µM. EGCG inhibited these marked inflammatory key signaling molecules by reducing the expression of phospho-nuclear factor-κB p65, -Akt, -ERK and -MAPK p38 while the total protein level of these signal proteins were not affected. In conclusion, the present findings suggested that EGCG possesses cardiomyocyte-protective action in reducing the LPS-induced inflammatory response due to the inhibition of the phosphorylation of Akt and ERK signaling molecules.

To explore the clinical effect and adverse reactions of Strychnos nux-vomica in bortezomib-induced peripheral neuropathy (BIPN) of patients with multiple myeloma (MM).A total of 19 MM patients with BIPN were enrolled and Nux Vomica Capsule (NVC, 0.4 g, thrice daily) were orally administrated for 30 days. Comparative analysis on parameters between pre- and post-therapy, including peripheral neuropathy (PN) grade, neurotoxicity score, Chinese medicine (CM) syndrome score, total neuropathy score (TNS), coagulation function, and serum nerve growth factor (NGF) levels were conducted. The adverse events were monitored.In BIPN of MM patients who received NVC, PN grade was lowered, neurotoxicity score was obviously decreased (P⩽0.01), and both CM syndrome score and TNS were remarkably decreased (P0.01). After the therapy, activated partial thromboplastin time was prolonged (P0.01) and fibrinogen was declined (P0.05), showing improvement in the hypercoagulable state of patients. No significant difference of NGF recovery degrees was detected between pre- and post-therapy (P0.05). No evident adverse reactions were observed during the course of treatment.Strychnos nux-vomica L. has significantly effect with a good safety in treatment of BIPN in MM patients.

Tooth enamel is composed of arrayed fluorapatite (FAP) or hydroxyapatite nanorods modified with Mg-rich amorphous layers. Although it is known that Mg2+ plays an important role in the formation of enamel, there is limited research on the regulatory role of Mg2+ in the synthesis of enamel-like materials. Therefore, we focus on the regulatory behavior of Mg2+ in the fabrication of biomimetic mineralized enamel-like structural materials. In the present study, we adopt a bioprocess-inspired room-temperature mineralization technique to synthesize a multilayered array of enamel-like columnar FAP/polymer nanocomposites controlled by Mg2+ (FPN-M). The results reveal that the presence of Mg2+ induced the compaction of the array and the formation of a unique Mg-rich amorphous-reinforced architecture. Therefore, the FPN-M array exhibits excellent mechanical properties. The hardness (2.42 ± 0.01 GPa) and Young's modulus (81.5 ± 0.6 GPa) of the as-prepared FPN-M array are comparable to those of its biological counterparts; furthermore, the enamel-like FPN-M array is translucent. The hardness and Young's modulus of the synthetic array of FAP/polymer nanocomposites without Mg2+ control (FPN) are 0.51 ± 0.04 and 43.5 ± 1.6 GPa, respectively. The present study demonstrates a reliable bioprocess-inspired room-temperature fabrication technique for the development of advanced high-performance composite materials.

RON (recepteur d'origine nantais) and MET (hepatocyte growth factor receptor) are tyrosine kinase receptors. Various cancers have aberrant RON and MET expression and activation, which contribute to cancer cell proliferation, invasiveness, and metastasis. Here, we explored RON and MET expression in pancreatic cancer and their relationship with overall survival (OS) time, and evaluated their significance as therapeutic targets of tyrosine kinase inhibitors in pancreatic cancer. We enrolled 227 patients with pancreatic cancer in the study. RON and MET expression was analyzed by immunohistochemical staining. Four human pancreatic cancer cell lines expressing variable levels of RON or MET and four MET superfamily inhibitors (BMS777607, PHA665752, INCB28060, Tivantinib) were used. The effect of the four tyrosine kinase inhibitors on cell viability, migration, and apoptosis were determined using cell viability, scratch wound healing, and Caspase-Glo 3/7 assays. Cellular signaling was analyzed by immunoprecipitation and western blotting. The therapeutic efficacy of the tyrosine kinase inhibitors was determined with mouse xenograft pancreatic cancer models in vivo. There was wide aberrant RON and MET expression in the cancer tissues. In 227 pancreatic cancer samples, 33% had RON overexpression, 41% had MET overexpression, and 15.4% had RON and MET co-overexpression. RON and MET expression were highly correlated. RON and MET expression levels were significantly related to OS. Patients with RON and MET co-overexpression had poorer OS. BMS777607 and PHA665752 inhibited pancreatic cancer cell viability and migration, and promoted apoptosis by inhibiting RON and MET phosphorylation and further inhibiting the downstream signaling pathways in vitro. They also inhibited tumor growth and further inhibited phosphorylated (phosphor)-RON and phospho-MET expression in the mouse xenograft models in vivo effectively. INCB28060, which inhibits the MET signaling pathway alone, was not effective. RON and MET can be important indicators of prognosis in pancreatic cancer. Tyrosine kinase inhibitors targeting RON and MET in pancreatic cancer are a novel and potential approach for pancreatic cancer therapy.

Learning from the process of biominerals formation provides tremendous ideas for developing advanced synthesis techniques. According to the structure formation of tooth enamel, a recombinant amelogenin his-AmelX was designed and constructed. The protein was over-expressed and could be conveniently purified in one-step heat treatment. The mineralization process of hydroxyapatite was initiated by enzyme AP and regulated by the recombinant amelogenin. Effects of solution pH value and mineralization duration were studied. It was demonstrated that his-AmelX could induce the nucleation of apatite and quicken the growth rate at pH 7.0-7.4, while impeded hydroxyapatite growth at pH 6.8. Moreover, a much denser layer of hydroxyapatite was achieved with the addition of his-AmelX. The present study may not only provide insight into the formation of natural biomaterials but also open a new path to prepare materials under environmentally benign conditions.

Background/Aims: To investigate the effects of bone marrow stromal cells (BMSCs) and underlying mechanisms in traumatic brain injury (TBI). Methods: Cultured BMSCs from green fluorescent protein-transgenic mice were isolated and confirmed. Cultured BMSCs were immediately transplanted into the regions surrounding the injured-brain site to test their function in rat models of TBI. Neurological function was evaluated by a modified neurological severity score on the day before, and on days 7 and 14 after transplantation. After 2 weeks of BMSC transplantation, the brain tissue was harvested and analyzed by microarray assay. And the coronal brain sections were determined by immunohistochemistry with mouse anti-growth-associated protein-43 kDa (anti-GAP-43) and anti-synaptophysin to test the effects of transplanted cells on the axonal regeneration in the host brain. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay and Western blot were used to detect the apoptosis and expression of BAX and BAD. Results: Microarray analysis showed that BMSCs expressed growth factors such as glial cell-line derived neurotrophic factor (GDNF). The cells migrated around the injury sites in rats with TBI. BMSC grafts resulted in an increased number of GAP-43-immunopositive fibers and synaptophysin-positive varicosity, with suppressed apoptosis. Furthermore, BMSC transplantation significantly downregulated the expression of BAX and BAD signaling. Moreover, cultured BMSC transplantation significantly improved rat neurological function and survival. Conclusion: Transplanted BMSCs could survive and improve neuronal behavior in rats with TBI. Mechanisms of neuroprotection and regeneration were involved, which could be associated with the GDNF regulating the apoptosis signals through BAX and BAD.

Ebolavirus (EBOV) life cycle involves interactions with numerous host factors, but it remains poorly understood, as does pathogenesis. Herein, we synthesized 65 siRNAs targeting host genes mostly connected with aspects of the negative-sense RNA virus life cycle (including viral entry, uncoating, fusion, replication, assembly, and budding). We produced EBOV transcription- and replication-competent virus-like particles (trVLPs) to mimic the EBOV life cycle. After screening host factors associated with the trVLP life cycle, we assessed interactions of host proteins with trVLP glycoprotein (GP), VP40, and RNA by co-immunoprecipitation (Co-IP) and chromatin immunoprecipitation (ChIP). The results demonstrate that RNAi silencing with 11 siRNAs (ANXA5, ARFGAP1, FLT4, GRP78, HSPA1A, HSP90AB1, HSPA8, MAPK11, MEK2, NTRK1 and YWHAZ) decreased the replication efficiency of trVLPs. Co-IP revealed nine candidate host proteins (FLT4, GRP78, HSPA1A, HSP90AB1, HSPA8, MAPK11, MEK2, NTRK1 and YWHAZ) potentially interacting with trVLP GP, and four (ANXA5, GRP78, HSPA1A, and HSP90AB1) potentially interacting with trVLP VP40. Ch-IP identified nine candidate host proteins (ANXA5, ARFGAP1, FLT4, GRP78, HSPA1A, HSP90AB1, MAPK11, MEK2 and NTRK1) interacting with trVLP RNA. This study was based on trVLP and could not replace live ebolavirus entirely; in particular, the interaction between trVLP RNA and host proteins cannot be assumed to be identical in live virus. However, the results provide valuable information for further studies and deepen our understanding of essential host factors involved in the EBOV life cycle.

Honokiol (HNK), an important bioactive compound purified from Magnolia officinalis Cortex, has been demonstrated to have manifold beneficial anti-oxidative, anti-inflammatory, anti-bacterial and antitumor pharmacological effects. In the present study, the association of HNK in the signaling mechanism associated with hydrogen peroxide (H2O2)-induced apoptosis of cultured mouse podocytes was investigated. HNK did not cause significant changes in podocyte viability when its concentration remained below 20 µM. MTS assay and flow cytometry confirmed that H2O2 significantly enhanced the rates of apoptosis while produce significant reduction in viability of podocytes. Following 24 h of pre-treatment with different concentrations of HNK, the viability of adherent podocytes increased and apoptosis significantly decreased in a dose-dependent manner below 20 µM. Reverse transcription-polymerase chain reaction and western blot results indicated that HNK significantly decreased the expression of mRNA and cleaved protein of caspase-3 and caspase-9 in podocytes pre-treated with H2O2. Furthermore, phosphorylation of the signaling molecules protein kinase B (Akt) and extracellular signal-regulated kinase (Erk) 1/2 appeared to increase following HNK treatment. In conclusion, HNK largely eliminated the role of promoting podocyte apoptosis in an oxidative stress environment, which was a protective factor on podocytes cultured with H2O2. The anti-oxidative stress mechanisms of HNK are partly due to suppressing the expression of caspase-3 and caspase-9 and upregulating phosphorylated-Akt and -Erk 1/2.

The aim of this study was to determine the prognostic factors and their significance in gastric cancer (GC) patients, using the artificial neural network (ANN) and Cox regression hazard (CPH) models. A retrospective analysis was undertaken, including 289 patients with GC who had undergone gastrectomy between 2006 and 2007. According to the CPH analysis, disease stage, peritoneal dissemination, radical surgery and body mass index (BMI) were selected as the significant variables. According to the ANN model, disease stage, radical surgery, serum CA19-9 levels, peritoneal dissemination and BMI were selected as the significant variables. The true prediction of the ANN was 85.3% and of the CPH model 81.9%. In conclusion, the present study demonstrated that the ANN model is a more powerful tool in determining the significant prognostic variables for GC patients, compared to the CPH model. Therefore, this model is recommended for determining the risk factors of such patients.

Aberrant expression of the RON receptor tyrosine kinase contributes to breast cancer malignancy. Although clinical trials of RON targeting are underway, the intriguing issue is the diversity of RON expression as evident by cancer cells expressing different variants including oncogenic RON160. The current study determines aberrant RON160 expression in breast cancer and its potential as a target for breast cancer therapy. Using mouse monoclonal antibody Zt/h12 in immunohistochemical staining of breast cancer tissue microarray, we observed that RON160 was expressed in high frequency in primary invasive ductal (77.2%, 61/79 cases), lobular (42.5%, 34/80 cases), and lymph node-involved (63.9%, 26/36 cases) breast cancer samples. Moreover, RON160 overexpression was predominantly observed in invasive ductal (26.6%, 21/79 cases) and lymph node-involved (33.3%, 12/36) cases. Among a panel of breast cancer cell lines analyzed, Du4475 cells naturally expressed RON160. Silencing RON160 expression by siRNA reduced Du4475 cell viability. Inhibition of RON160 signaling by tyrosine kinase inhibitor PHA665752 also suppressed Du4475 cell anchorage-independent growth and induced apoptotic cell death. Studies in vivo revealed that PHA665752 inhibited 3T3- RON160 and Du4475 cell-mediated tumor growth in mouse mammary fat pad. A 60% reduction in tumor volume compared to controls was achieved after a 13-day treatment. We conclude from these studies that RON160 is highly expressed in breast cancer and its signaling is integrated into cellular signaling network for tumor cell growth and survival. Experimental treatment by PHA665752 in Du4475 breast cancer xenograft model highlights the significance of RON160 as a drug target in molecular-targeted breast cancer therapy.

Cardiovascular toxicity is a major challenge for the pharmaceutical industry and predictive screening models to identify and eliminate pharmaceuticals with the potential to cause cardiovascular toxicity in humans are urgently needed. In this study, taking advantage of the transparency of larval zebrafish, Danio rerio, we assessed cardiovascular toxicity of seven known human cardiotoxic drugs (aspirin, clomipramine hydrochloride, cyclophosphamide, nimodipine, quinidine, terfenadine and verapamil hydrochloride) and two non-cardiovascular toxicity drugs (gentamicin sulphate and tetracycline hydrochloride) in zebrafish using six specific phenotypic endpoints: heart rate, heart rhythm, pericardial edema, circulation, hemorrhage and thrombosis. All the tested drugs were delivered into zebrafish by direct soaking and yolk sac microinjection, respectively, and cardiovascular toxicity was quantitatively or qualitatively assessed at 4 and 24 h post drug treatment. The results showed that aspirin accelerated the zebrafish heart rate (tachycardia), whereas clomipramine hydrochloride, cyclophosphamide, nimodipine, quinidine, terfenadine and verapamil hydrochloride induced bradycardia. Quinidine and terfenadine also caused atrioventricular (AV) block. Nimodipine treatment resulted in atrial arrest with much slower but regular ventricular heart beating. All the tested human cardiotoxic drugs also induced pericardial edema and circulatory disturbance in zebrafish. There was no sign of cardiovascular toxicity in zebrafish treated with non-cardiotoxic drugs gentamicin sulphate and tetracycline hydrochloride. The overall prediction success rate for cardiotoxic drugs and non-cardiotoxic drugs in zebrafish were 100% (9/9) as compared with human results, suggesting that zebrafish is an excellent animal model for rapid in vivo cardiovascular toxicity screening. The procedures we developed in this report for assessing cardiovascular toxicity in zebrafish were suitable for drugs delivered by either soaking or microinjection.

Background Spinal cord injury (SCI) is a serious disease which can lead to bad consequence in patients. Gene therapies, as an effective strategy, have been developed for the treatment of several diseases. But the effect for the treatment of SCI is also waiting to be practiced. Objective Here, we explored the effect of NGF administration carried by herpes simplex virus (HSV) in the injured spinal cord. Methods Transgenic recombinant containing human NGF was constructed by using pSP72 plasmid, then enveloped by non-replication HSV vector with deleted ICP27, ICP4 and ICP34.5 genes. Next, HSV recombinant carrying NGF was injected into cerebrospinal fluid in the lumbar cord to detect the effect of NGF for the improvement of motor function, indicated by BBB score. Meanwhile, IHC, QPCR and WB were used to confirm the NGF transduction. Results After SCT, BBB score was largely decreased, followed by a gradual limit recovery with time going on. Q-PCR confirmed that the mRNA expression of NGF was increased in the spinal cord at 28 days post-operation, compared with that in the sham group, which suggests endogenous NGF may be available to the limit repair of motor function. Moreover, HSV carried NGF was injected into subarachnoid space of the spinal cord, which results in a significant functional improvement in hindlimbs from 7dpo to 49dpo. The level of NGF in HSV-NGF administrated group was obviously higher than that in the empty vector group and SCT group, only. Conclusion Our results demonstrate that releasing of HSV-NGF-recombinant in subarachnoid space, can effectively improve the motor function in hindlimbs of rats subjected to SCT, which supports that strategy of HSV carrying NGF may be used for the treatment of SCI in future clinic practice.

Spinal cord injury (SCI) often causes severe functional impairment with poor recovery. The treatment, however, is far from satisfaction, and the mechanisms remain unclear. By using proteomics and western blot, we found spinal cord transection (SCT) resulted in a significant down-regulation of α-synuclein (SNCA) in the motor cortex of SCT rats at 3 days post-operation. In order to detect the role of SNCA, we used SNCA-ORF/shRNA lentivirus to upregulate or knockdown SNCA expression. In vivo, SNCA-shRNA lentivirus injection into the cerebral cortex motor area not only inhibited SNCA expression, but also significantly enhanced neurons' survival, and attenuated neuronal apoptosis, as well as promoted motor and sensory function recovery in hind limbs. While, overexpression SNCA exhibited the opposite effects. In vitro, cortical neurons transfected with SNCA-shRNA lentivirus gave rise to an optimal neuronal survival and neurite outgrowth, while it was accompanied by reverse efficiency in SNCA-ORF group. In molecular level, SNCA silence induced the upregulation of Bcl-2 and the downregulation of Bax, and the expression of NGF, BDNF and NT3 was substantially upregulated in cortical neurons. Together, endogenous SNCA play a crucial role in motor and sensory function regulation, in which, the underlying mechanism may be linked to the regulation of apoptosis associated with apoptotic gene (Bax, Bcl2) and neurotrophic factors expression (NGF, BDNF and NT3). These finds provide novel insights to understand the role of SNCA in cerebral cortex after SCT, and it may be as a novel treatment target for SCI repair in future clinic trials.

Human cytomegalovirus glycoprotein B (gB) represents a target for diagnosis and treatment in view of the role it plays in virus entry and spread. Nevertheless, to our knowledge, rare detection of a gB antigen has been reported in transplant patients and limited information is available about diagnostic gB monoclonal antibodies (mAbs). Our aim was to develop gB mAbs with diagnostic potential. Hydrophilic gB peptides (ST: amino acids 27-40, SH: amino acids 81-94) of favorable immunogenicity were synthesized and used to immunize BALB/c mice. Two mAbs, named ZJU-FH6 and ZJU-FE6, were generated by the hybridoma technique and limited serial dilution and then characterized by indirect ELISA, Western blotting, immunoprecipitation, and immunohistochemical staining. The mAbs displayed high titers of specific binding affinities for the ST and SH synthetic peptides at an mAb dilution of 1:60,000 and 1:240,000, respectively. Western blotting and immunoprecipitation indicated that these mAbs recognized both denatured and native gB of the Towne and AD169 strains. The mAbs, when used as the primary antibody, showed positive staining in cells infected with both Towne and AD169 strains. The mAbs were then tested on patients submitted to allogeneic hematopoietic stem cell transplantation. The gB antigen positivity rates of the patients tested using ZJU-FH6 and ZJU-FE6 were 62.0 and 63.0%, respectively. The gB antigen showed a significant correlation with the level of pp65 antigen in peripheral blood leukocytes. In conclusion, two potential diagnostic gB mAbs were developed and were shown to be capable of recognizing gB in peripheral blood leukocytes in a reliable manner.

The best characterized signalling pathway employed by transforming growth factor-beta (TGF-β) is the Smad pathway. We focused on Smad4, because it is essential for the activation of Smad-dependent target genes. We aimed to explore the possibility of inhibiting scar formation after wounding by blocking TGF-β signalling by means of a gene therapy approach using adenovirus-mediated expression of mutant Smad4. The coding sequence of the dominant-negative mutant Smad4ΔM4, which has a deletion in the linker region of Δ275–322, was introduced by homologous recombination into an adenovirus vector to generate the recombinant vector Ad-ΔM4, which encoded Smad4ΔM4. Mouse fibroblast NIH 3T3 cells were transfected with Ad-ΔM4 and cell proliferation, collagen protein production, and the expression of collagen type I and type III mRNA were evaluated in vitro using a cell proliferation test, western blot analysis, and RT-PCR, respectively. Cell proliferation and the expression of collagen type I and type III mRNA and protein were all inhibited by the transfection of Ad-ΔM4. In vivo, Ad-ΔM4 was applied externally to wounds on rats, and histological examination and quantification of the scars were performed to evaluate the curative effect. The transfection of Ad-ΔM4 successfully inhibited scar formation in rat wounds. In conclusion, Ad-ΔM4 can block the TGF-β signalling of mouse wound cells effectively. In addition, gene therapy with Ad-ΔM4 can effectively inhibit wound scarring in rats and may potentially be applied to clinical treatment of scars.

To evaluate the regulatory effects of honokiol on high-glucose (HG)-induced inflammatory responses of human renal mesangial cells (HRMCs).We performed MTS assays to determine the non-cytotoxic concentration of honokiol for HRMCs. Enzyme-linked immunosorbent assays were performed to analyze the expressions of the proteins interleukin (IL)-1β, IL-18, tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-β1, monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1α, RANTES, and prostaglandin (PG) E2. The total nitric oxide (NO) concentration was determined using the Griess reaction.Treatment with 50 mmol/L glucose markedly increased the level of IL-1β, IL-18, TNF-α, PGE2, NO, TGF-β1, MCP-1, MIP-1α, and RANTES. Honokiol (~20 μmol/L) treatment inhibited the HG-induced expression of inflammatory cytokines such as IL-1β, IL-18, TNF-α, PGE2, NO, and TGF-β1 in a dose-dependent manner. Moreover, it markedly inhibited the expression of chemokines such as MCP-1, MIP-1α, and RANTES, which are upregulated under HG conditions.Honokiol inhibits the HG-induced expression of inflammatory factors in HRMCs. Honokiol may be considered a promising drug with potent anti-inflammatory activities in addition to its strong anti-cancer, anti-angiogenesis, and anti-neurodegenerative effects.

Overexpression of the RON receptor tyrosine kinase exists in various cancers and contributes to malignant progression. To validate RON as a targeting moiety for delivery of chemoagents for enhanced tumor cytotoxicity, immunoliposomes (IL) loaded with doxorubicin (Dox) were formulated followed by postinsertion of monoclonal antibodies Zt/g4, Zt/c1, or their Fab fragments specific to the RON extracellular domains. Flow cytometry analysis showed that Zt/g4 or Zt/c1-IL binds to cancer cells and causes RON internalization as evident in confocal analysis of intracellular fluorescence intensity. The antibody-directed IL uptake by cancer cells is in both dose and time-dependent manners. Studies of cytotoxicity of individual IL in vitro against colon or breast cancer cell lines revealed that Zt/g4 directed Dox-IL displayed increased cytotoxic activities with a significant reduction of IC(50) values. An average of 8-fold increases in cytotoxic efficiency was achieved among four cell lines tested. Moreover, Zt/g4 directed Dox-IL also displayed the effective killing of cancer cells that are insensitive to pegylated liposomal doxorubicin. The effect of Zt/c1-Dox-IL was not as strong as Zt/g4-Dox-IL, and only moderate activities were observed. IL coupled with the Fab fragments of Zt/g4 or Zt/c1 show moderate activities against cancer cells. The ineffectiveness seemed to be related to the weak activities of the Fab fragments in the induction of RON internalization, which resulted in reduced drug uptakes. We conclude that anti-RON antibody-directed drug delivery is effective for increased uptake of cytotoxic drugs. Antibody-based RON targeting could be developed into a potential therapeutic for treatment of malignant cancers.

The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 is a vaccine immunogen that has been studied extensively. To enhance the immune response of cells against HIV-1 gp120, we tested the coexpression of gp120N with interferon-gamma (IFN-gamma) as an immune adjuvant. Two recombinant prokaryotic plasmids were constructed: the pET44b-HIV-1-gp120N plasmid construct carried the HIV-1 gp120N gene (pET44-gp120N), whereas the pET44b-HIV-1-gp120N-IFN-gamma plasmid construct carried a fusion gp120N-IFN-gamma gene (pET44b-gp120N-IFN-gamma). Target protein expression was achieved in E. coli BL21 (DE3) cells by chemical induction. To test the immunological activity of the proteins, mice were injected with a control, gp120N, or the fusion gp120N-IFN-gamma protein. The serum and spleen cells of the mice were collected for immunological detection. Results showed that specific T lymphocyte proliferation and the expression of the Th1-type cytokines (IL-2 and IFN-gamma) were higher in the gp120N-IFN-gamma group than the other two groups (P0.05). No difference was observed in the expression levels of the Th2-type cytokines (IL-4 and IL-10; P0.05). These results suggest that IFN-gamma plays a prominent role as an immune adjuvant when coexpressed with HIV-1 gp120N. IFN-gamma enhances the specific cell immune response of mice against HIV-1 gp120.

Aim: The aim of the present study was to investigate the expression of Toll-like receptors (TLR) 7 and 9 in peripheral blood mononuclear cells (PBMC) of patients with chronic hepatitis B virus (HBV) infection and related hepatocellular carcinoma. Methods: The study group was comprised of 52 patients: 41 with chronic hepatitis B and 11 healthy controls. The protein and mRNA levels of TLR7 and TLR9 were evaluated using real-time PCR, Western blot analysis, and flow cytometry. We also detected the serum viral load of HBV in the patients and analyzed the correlation between HBV–DNA copies and the TLR expression. Results: Our results demonstrated a lower TLR7 expression in all HBV infection groups compared to the controls. We found that HBV infection led to a decreased expression of TLR9 mRNA, but an increased expression of the TLR9 protein compared to the healthy group. The TLR protein levels are related to serum HBV–DNA ( P Conclusion: There are downregulations of TLR7 expression and TLR9 mRNA in PBMC of HBV-infected patients, but an increased TLR9 expression at the protein level.

Claudins are integral membrane proteins essential in tight junction formation and function. Altered expression of claudins has been implicated in epithelial malignant transformation. We report here that activation of recepteur d'origine nantais (RON) differentially regulates tight junction function and claudin expression. In Martin-Darby canine kidney (MDCK) cells, macrophage-stimulating protein-induced RON activation or expression of constitutively active variant RON160 significantly disrupted cellular tight junctions and reduced transepithelial electrical resistance. These changes were featured by diminished claudin-1 expression and redistribution of claudin-3 and -4 into cytoplasmic compartments. The inhibition of claudin-1 was also seen in breast cancer T-47D cells. By analyzing the signaling events, we found that activation of the extracellular signal-regulated kinase 1/2 pathway is required for RON-mediated inhibition of claudin-1 expression and redistribution of claudin-3 and -4. Results from luciferase reporter assays showed that inhibition is acted at the transcriptional levels because RON activation decreases claudin-1 promoter activities and increases transcriptional repressor Snail-1 expression. Functional analysis further revealed that reduced claudin-1 expression is linked to increased motilities of MDCK and T-47D cells as evident in cell migration and wound-healing assays. Forced expression of claudin-1 prevented RON-mediated cell migration and restored cell morphologies to their original epithelial appearance. In conclusion, RON activation differentially regulates claudin expression in epithelial cells. Inhibition of claudin-1 expression may represent a novel mechanism that contributes to RON-mediated invasive activities, leading to increased tumor malignancy.

BACKGROUND Despite low morbidity, acute or subacute spinal epidural hematoma may develop quickly with a high tendency to paralysis. The delay of diagnosis and therapy often leads to serious consequences. In this study we evaluated the effects of a series of methods for the diagnosis and treatment of the hematoma in 11 patients seen in our hospital. METHODS Of the 11 patients (8 males and 3 females), 2 had the hematoma involving cervical segments, 2 cervico-thoracic, 4 thoracic, 1 thoraco-lumbar, and 2 lumbar. Three patients had quadriplegia, including one with central cord syndrome; another had Brown-Sequard's syndrome; and the other seven had paraplegia. Five patients were diagnosed at our hospitals within 3 - 48 hours after appearance of symptoms, and 6 patients were transferred from community hospitals within 21 - 106 hours after development of symptoms. Key dermal points, key muscles and the rectal sphincter were determined according to the American Spinal Injury Society Impairment Scales as scale A in two patients, B in 5 and C in 4. Emergency MRI in each patient confirmed that the dura mater was compressed in the spinal canal, with equal intensity or hyperintensity on T(1) weighted image and mixed hyperintensity on T(2) weighted image. Preventive and curative measures were taken preoperatively and emergency operation was performed in all patients. Open laminoplasty was done at the cervical and cervico-thoracic segments, laminectomy at the thoracic segments, laminectomy with pedicle screw fixation at the thoraco-lumbar and lumbar segments involving multiple levels, and double-sided laminectomy with the integrity of articular processes at the lumbar segments involving only a single level. During the operation, special attention was given to hematoma evacuation, hemostasis and drainage tube placement. RESULTS Neither uncontrollable hemorrhage nor postoperative complications occurred. All patients were followed up for 1 - 6 years. A marked difference was noted between postoperative and preoperative scales (u = 3.66, P < 0.01). Most patients recovered after therapy, but the recovery of patients treated at our hospitals was superior to that of those transferred from community hospitals (t = 2.95, P < 0.05). Of the patients treated at our hospitals, 4 were cured and 1 was upgraded with scale from A to D, whereas none of those transferred from community hospitals recovered completely, even one remained scale C. CONCLUSIONS Physical examination plus MRI is essential to early diagnosis of acute or subacute spinal epidural hematoma. Preventive and curative measures including emergency operation are helpful to the recovery of patients' nerve function.

Spinal cord injury (SCI), a severe health problem in worldwide, was commonly associated with functional disability and reduced quality of life. As the expression of brain-derived neurotrophic factor (BDNF) was substantial event in injured spinal cord, we hypothesized whether BDNF-overexpression could be in favor of the recovery of both sensory function and hindlimb function after SCI. By using BDNF-overexpression transgene mice [CMV-BDNF 26 (CB26) mice] we assessed the role of BDNF on the recovery of neurological behavior in spinal cord transection (SCT) model. BMS score and tail-flick test was performed to evaluate locomotor function and sensory function, respectively. Immunohistochemistry was employed to detect the location and the expression of BDNF, NeuN, 5-HT, GAP-43, GFAP as well as CGRP, and the level of p-AKT and AKT were examined through western blot analysis. BDNF overexpressing resulted in significant locomotor functional recovery from 21 to 28 days after SCT, compared with wild type (WT)+SCT group. Meanwhile, the NeuN, 5-HT and GAP-43 positive cells were markedly increased in ventral horn in BDNF overexpression animals, compared with WT mice with SCT. Moreover, the crucial molecular signal, p-AKT/AKT has been largely up-regulated, which is consistent with the improvement of locomotor function. However, in this study, thermal hyperpathia encountered in sham (CB26) group and WT+SCT mice and further aggravated in CB26 mice after SCT. Also, following SCT, the significant augment of positive-GFAP astrocytes and CGRP fibers were found in WT+SCT mice, and further increase was seen in BDNF over-expression transgene mice. BDNF-overexpression may not only facilitate the recovery of locomotor function via AKT pathway, but also contributed simultaneously to thermal hyperalgesia after SCT.

Cancer stem cells (CSCs) contribute to pancreatic cancer tumorigenesis through tumor initiation, drug resistance, and metastasis. Currently, therapeutics targeting pancreatic CSCs are under intensive investigation. This study tested a novel strategy that utilizes the RON receptor as a drug delivery moiety for increased therapeutic activity against pancreatic CSCs. CD24(+)CD44(+)ESA(+) triple-positive pancreatic CSCs (CSCs(+24/44/ESA)) were obtained from spheroids of pancreatic L3.6pl cancer cells by sequential magnetic cell sorting methods. These cells displayed a spherical growth pattern, expressed the unique self-renewal marker Bmi-1, redifferentiated into an epithelial phenotype, acquired an epithelial to mesenchymal phenotype, and caused tumor formation in animal models. Among several receptor tyrosine kinases examined, RON was highly expressed and sustained by CSCs(+24/44/ESA). This feature provided the cellular basis for validating the therapeutic effectiveness of anti-RON antibody Zt/c9-directing doxorubicin-immunoliposomes (Zt/c9-Dox-IL). Zt/c9-Dox-IL specifically interacted with CSCs(+24/44/ESA) and rapidly caused RON internalization, which led to the uptake of liposome-coated Dox. Moreover, Zt/c9-Dox-IL was effective in reducing viability of L3.6pl cells and CSCs(+24/44/ESA). The IC(50) values between free Dox (62.0 ± 3.1 μM) and Zt/c9-Dox-IL (95.0 ± 6.1 μM) treated CSCs(+24/44/ESA) were at relatively comparable levels. In addition, Zt/c9-Dox-IL in combination with small molecule inhibitors lapatinib, sunitinib, or dasatinib further reduced the viability of CSCs(+24/44/ESA). In conclusion, RON expression by CSCs(+24/44/ESA) is a suitable molecule for the targeted delivery of chemoagents. The anti-RON antibody-directed delivery of chemotherapeutics is effective in reducing viability of pancreatic CSCs.

Hypoxia contributes to acquired drug resistance in various cancer cells. The underlying mechanism is cellular insensitivity regulated by hypoxia-inducible factors (HIF), which impairs drug uptake, transport, and metabolism. The current study determines anti-RON antibody-directed cytotoxicity of doxorubicin (Dox)-immunoliposomes (IL) in hypoxic colon cancer cells.Cells were cultured under hypoxia (1% O(2), 5% CO(2), and 96% N(2)) for 24 h. Dox-loaded IL were formulated followed by post-insertion of monoclonal antibody Zt/g4 specific to RON. Western blotting was used to detect HIF-1α and RON expression. Cellular uptake of Zt/g4-conjugated IL was determined by confocal and internalization assays. Cell viability was assessed by the MTT assay.RON and HIF-1α expression were observed in hypoxic colon HCT116 and SW620 cells. Resistance to Dox-induced cytotoxicity was acquired in hypoxic cells with increased IC(50) values. However, acquired resistance was attenuated by Zt/g4-directed Dox-IL, which displays increased cytotoxic activities. IL binding and uptake revealed that hypoxic RON expression is functional, which mediates high levels of Zt/g4-Dox-IL binding and cytoplasmic internalization. Zt/g4-Dox-IL is effective in killing hypoxic HCT116 and SW620 cells with reduced IC(50) values compared to Dox and pegylated-liposomal Dox. These effects were dependent on hypoxic RON expression. HCC1937 cells with diminished RON expression under hypoxia were insensitive to Zt/g4-Dox-IL-induced cytotoxic effect.RON expressed by hypoxic colon cancer cells is thus a potential targeting molecule for delivery of chemotherapeutics. The ability of anti-RON mAb to direct Dox-IL cytotoxicity could be developed for attenuating hypoxia-acquired drug resistance in various cancer cells.

Objective Allicin is believed to be the main component responsible for the biological activity of garlic. The regulation of cell apoptosis may have therapeutic potential for trauma/hemorrhagic shock, and previous studies have demonstrated that allicin exerts protective effects against tissue ischemia-reperfusion injury. Therefore, this study examined the effects of allicin on apoptosis-related organ damage, induced by trauma/hemorrhagic shock. Methods Studies were performed on an in vivo model of spontaneously breathing rats with induced trauma/hemorrhagic shock; the left lower lobe of the lungs, left kidney, and intestine were resected, and the rats were subjected to femur fracture, ischemia for 30 mins, and reperfusion for 20 mins. Allicin (30 microg/kg) was administered during reperfusion. Results Allicin administered during reperfusion markedly attenuated injury and apoptosis of the lungs, kidneys, and intestine and decreased the concentrations of lactic acid and creatinine, the number of in situ TdT-mediated dUTP nick-end labeling-positive cells, and the activity and expression of caspase-3 and -9 (as determined by Western blot). Furthermore, immunohistochemistry and Western blot performed 24 hrs after reperfusion revealed increases in the levels of nuclear factor kappaB, phosphorylated p38, and extracellular signal-regulated kinase 1 mitogen-activated protein kinase in the allicin-untreated group when compared with the sham rats. Allicin treatment downregulated the levels of nuclear factor kappaB and phosphorylated p38 mitogen-activated protein kinase but did not modify those of phosphorylated extracellular signal-regulated kinase 1 mitogen-activated protein kinase. Conclusion Allicin attenuates tissue injury and inhibits trauma/hemorrhagic shock- and reperfusion-induced apoptosis in several important organs via the p38 mitogen-activated protein kinase signal transduction pathway that functions to stimulate the activation of nuclear factor-kappaB, caspase-3 and -9, but not of extracellular signal-regulated kinase 1 mitogen-activated protein kinase.

To investigate part mechanisms of Th cell differentiation, and to observe the interference effect of Qingxin-II Recipe in the chronic stage of viral myocarditis (VMC), so as to provide some experimental evidences for illuminating the pathogenesis of VMC and treatment mechanisms of Qingxin-II Recipe.According to 20%-40% death rate of experiment in advance, 100 BALB/c male mice (4 weeks old and weighing 12-15 g) were used. Twenty mice were randomly assigned to normal control group, and the other 80 mice were intraperitoneally injected with 0.1 ml normal saline containing coxsackie virus B3 at the 1st, 4th and 28th day (the virus densities were 1:2000, 1:1000 and 1:500 respectively) to induce chronic VMC. At the 42nd day, the surviving mice were randomly divided into untreated group and treatment group, with 20 mice in each group. Mice in the treatment group were orally administered with 0.2 ml Qingxin-II Recipe every day, while mice in the normal control group and the untreated group were administered with 0.2 ml normal saline. All the mice were sacrificed after 45 days, and the sera, heart and spleen cells were collected. Then the myocardial pathological changes were observed by using a light microscope, and the levels of IL-4, IL-10 and IFN-gamma in serum were detected by enzyme linked immunosorbent assay (ELISA). And the Th cell differentiation was observed by flow cytometry.No obvious myocardial pathological changes were observed in mice of the normal control group. Myocardial pathological changes in the treatment group were slighter than those in the untreated group. The difference of serum IL-10 level between the normal control group and the untreated group showed no significance (P0.05), and the levels of IFN-gamma and IL-4 of the untreated group were higher than those of the normal control group (P0.05 or P0.01). There was no statistical difference in IL-10 level between the treatment group and the untreated group (P0.05), while the serum levels of IL-4 and IFN-gamma of the treatment group were lower than those of the untreated group (P0.05 or P0.01). There was no significant difference of the Th1 cell responder between the treatment group and the untreated group (P0.05), while the Th2 cell responder was inhibited significantly in the treatment group (P0.05).Qingxin-II Recipe can restore the balance of Thl and Th2 cells through inhibiting the reaction of Th2.

AIM: To evaluate the inhibitory effects of DNAzymes on the expressions of hepatitis B virus (HBV) s (HBsAg) and e (HBeAg) in 2.2.15 cells, and to explore the potential therapeutic effects of DNAzymes on replication of HBV genome. METHODS: DNAzymes DrzBS and DrzBC specific to HBV (ayw subtype) s gene ORF A157UG and e gene ORF A1816UG, were designed and synthesized. Inhibitory effects of DrzBS or DrzBC on the expressions of HBV s and e genes as well as HBV DNA levels in culture supernatants were observed in 2.2.15 cells. RESULTS: After being treated with DrzBS or DrzBC, the expression of HBV s or e genes in 2.2.15 cells was depressed dramatically. The maximum inhibition rate was 94.2% and 91.8% for DrzBS and DrzBC, respectively. The concentration for effective inhibition of both DrzBS and DrzBC was within 0.1-2.5 µmol/L, showing a dose-dependence. The efficiency of inhibiting HBsAg, HBeAg in 2.2.15 cells by DrzBS or DrzBC was higher than that of the same target genes by antisense oligonucleotides (ASON). The concentration for effective inhibition of DNAzymes was at least 10-fold lower compared with ASON controls. Neither inhibition on the replication of HBV DNA nor toxicity to 2.2.15 cells was observed. CONCLUSION: DrzBS and DrzBC can block the expression of HBV s- and e-genes in 2.2.15 cells and provide a specific and effective anti-HBV gene therapeutic means.

Objective: To construct a cDNA library from human liver tissue with chronic hepatitis B and check its quality for investigating the expression level of liver tissue infected by hepatitis B virus. This will then be used to find the relevant genes and interesting proteins associated with the development of hepatitis B. Methods: The total RNA from liver tissue with chronic hepatitis B was extracted and the mRNA was purified using TRIZOL method. Switching mechanism at 5′ end of the RNA transcript (SMART) technique and CDS III/3′ primer were used for first-strand cDNA synthesis. Long distance polymerase chain reaction (LD PCR) was then used to synthesize the double-strand cDNA that was then digested by Sfi I and fractionated by CHROMA SPIN-400 column. The longer than 0.4 kb cDNAs were collected and ligated to λTriplEx2 vector. Then λ phage packaging reaction and library amplification were performed. The qualities of both unamplified and amplified cDNA libraries were strictly checked by conventional titer determination. Fourteen plaques were randomly picked and tested using PCR with universal primers derived from the sequence flanking the vector. Results: The titers of unamplifed and amplified libraries were 1.94×106 pfu/ml and 1.49×109 pfu/ml respectively. The percentages of recombinants from both libraries were 98.15% in unamplified library and 98.76% in amplified library. The lengths of the inserts were 1.23 kb in average, 1–2 kb in 64.29%, and 0.5–1.0 kb in 35.71%. Conclusion: A high quality cDNA library from human liver tissue with chronic hepatitis B was successfully constructed.

Objectives: To evaluate the inhibitory effect mediated by combination of small interfering RNAs (siRNAs) targeting different sites of hepatitis B virus (HBV) transcripts on the viral replication and antigen expression in vitro. Methods: (1) Seven siRNAs targeting surface (S), polymerase (P) or precore (PreC) region of HBV genome were designed and chemically synthesized. (2) HBV-producing HepG2.2. 15 cells were treated with or without siRNAs for 72 h. (3) HBsAg and HBeAg in the cell culture medium were detected by enzyme-linked immunoadsorbent assay. (4) Intracellular viral DNA was quantified by real-time PCR (Polymerase Chain Reaction). (5) HBV viral mRNA was reverse transcribed and quantified by real-time PCR. (6) The change of cell cycle and apoptosis was determined by flow cytometry. Results: Our data demonstrated that synthetic small interfering RNAs (siRNAs) targeting S and PreC gene could efficiently and specifically inhibit HBV replication and antigen expression. The expression of HBsAg and HBeAg and the replication of HBV could be specifically inhibited in a dose-dependent manner by siRNAs. Furthermore, our results showed that the combination of siRNAs targeting various regions could inhibit HBV replication and antigen expression in a more efficient way than the use of single siRNA at the same final concentration. No apoptotic change was observed in the cell after siRNA treatment. Conclusion: Our results demonstrated that siRNAs exerted robust and specific inhibition on HBV replication and antigen expression in a cell culture system and combination of siRNAs targeting different regions exhibited more potency.

AIM: To investigate the role of human La protein in HBV mRNA expression. METHODS: Three human La protein (hLa) specific siRNA expression cassettes (SECs) containing U6+1 promoter were prepared via one-step overlapping extension PCR. After transfection with SECs into HepG2 cells, inhibition effects on hLa expression were analyzed by semi-quantitative RT-PCR and Western blotting. Then, effective SECs were screened out and transfected into 2.2.15 cells, a stable HBV-producing cell line. HBV surface antigen (HBsAg) and e antigen (HBeAg) secretions into culture media were detected by microparticle enzyme immunoassay (MEIA) and HBs and HBe mRNA levels were analyzed by semi-quantitative RT-PCR. RESULTS: SEC products containing U6+1 snRNA promoter, and 3 sites of hLa mRNA specific siRNA were obtained successfully by one-step overlapping extension PCR and could be directly transfected into HepG2 cells, resulting in inhibition of La protein expression in both mRNA and protein levels, among which U6+1-hLa833 was the most efficient, which reduced 18.6-fold mRNA and 89% protein level respectively. In 2.2.15 cells, U6+1-hLa833 was also efficient on inhibition of hLa expression. Furthermore, semi-quantitative RT-PCR showed that HBs and HBe mRNA levels were significantly decreased by 8- and 66-fold in U6+1-hLa833 transfected cells compared to control. Accordingly, HBsAg and HBeAg secretions were decreased partly posttransfection with SECs. CONCLUSION: PCR-based SECs can be used to mediate RNAi in mammalian cells and provide a novel approach to study the function of La protein. The inhibition of La protein expression can result in a significant decrease of HBV mRNA, which implies that the hLa protein is also involved HBV RNA metabolism as one of the HBV RNA-stabilizing factors in human cells.

Thymosin alpha 1 (Talpha1) has immunomodulatory effects on T-cells, NK-cells and macrophages, but its action on dendritic cells (DCs), which are recognized as the sole professional antigen presenting cells (APCs) capable of priming naïve T-cells, is poorly understood. In this study, the effect of Talpha1 in vitro on murine bone marrow-derived DCs (BMDCs) maturation, differentiation, and function with or without tumor necrosis factor-alpha (TNF-alpha), which is one of the important inflammatory parameters, has been investigated. We have shown, through flow cytometry, ELISA and mixed leukocyte reaction (MLR), that Talpha1 promoted CD4-expressed DC differentiation and the expression of activation markers, but did not influence IL-12 production and T cell-stimulatory capacity of DCs in the absence of TNFalpha during BMDCs maturation. Furthermore, in the presence of TNF-alpha, Talpha1 has been shown not only to promote the expression of CD4 on MHC class II+ DCs and enhance the up-regulated levels of mature markers induced by TNF-alpha, but also to suppress the up-regulated IL-12 production. Particularly, thus effects seen were obvious at pharmacological Talpha1 concentrations. However, Talpha1 did not inhibit TNF-alpha-induced T-cell stimulating function of DCs. This is the first reported example of a direct Talpha1-DC interaction and suggests a mechanism by which Talpha1 may in part affect T-cell responses by acting at the DC level and it may play an important role in the modulation of the local inflammatory responses in vivo.

Background Overexpression of the RON receptor tyrosine kinase contributes to epithelial cell transformation, malignant progression, and acquired drug resistance. RON also has been considered as a potential target for therapeutic intervention. This study determines biochemical features and inhibitory activity of a mouse monoclonal antibody (mAb) Zt/f2 in experimental cancer therapy. Results Zt/f2 is a mouse IgG2a mAb that is highly specific and sensitive to human RON and its oncogenic variants such as RON160 (ED50 = 2.3 nmol/L). Receptor binding studies revealed that Zt/f2 interacts with an epitope(s) located in a 49 amino acid sequence coded by exon 11 in the RON β-chain extracellular sequences. This sequence is critical in regulating RON maturation and phosphorylation. Zt/f2 did not compete with ligand macrophage-stimulating protein for binding to RON; however, its engagement effectively induced RON internalization, which diminishes RON expression and impairs downstream signaling activation. These biochemical features provide the cellular basis for the use of Zt/f2 to inhibit tumor growth in animal model. Repeated administration of Zt/f2 as a single agent into Balb/c mice results in partial inhibition of tumor growth caused by transformed NIH-3T3 cells expressing oncogenic RON160. Colon cancer HT-29 cell-mediated tumor growth in athymic nude mice also was attenuated following Zt/f2 treatment. In both cases, ~50% inhibition of tumor growth as measured by tumor volume was achieved. Moreover, Zt/f2 in combination with 5-fluorouracil showed an enhanced inhibition effect of ~80% on HT-29 cell-mediated tumor growth in vivo. Conclusions Zt/f2 is a potential therapeutic mAb capable of inhibiting RON-mediated oncogenesis by colon cancer cells in animal models. The inhibitory effect of Zt/f2 in vivo in combination with chemoagent 5-fluorouracil could represent a novel strategy for future colon cancer therapy.

OBJECTIVE To study the inhibitory effect of Kangxian Recipe (KXR) on TGF-beta 1 induced hepatocyte apoptosis. METHODS The in vitro model of hepatocyte apoptosis was established by cell biologic methods, utilizing the characteristics of TGF-beta 1 to observe the inhibitory effect of KXR on hepatocyte apoptosis. RESULTS TGF-beta 1 induced apoptosis of hepatocyte in a dose-dependent manner. The apoptosis rate of 2.2.15 cells was 63% when 500 ng/L TGF-beta 1 was used, while for HepG2 cell, it was merely 44%. After treatment of 20 ng/L KXR, the apoptosis rate of the two kinds of cell lines lowered to 33% and 24% respectively. The inhibition rate of both groups was about 50%. CONCLUSION KXR had strong inhibitory effect on hepatocyte apoptosis induced by TGF-beta 1.