Search

Objective: To evaluate the therapeutic effect of nasal coblation plasma surgery for the treatment of persistent allergic rhinitis (PAR)., Methods: One hundred patients with mite-sensitized moderate to severe PAR who underwent nasal coblation plasma surgery (inferior turbinoplasty plus nasal agger ablation) were enrolled in this study. There were 68 male and 32 female patients aged 16 to 62 years (mean, 36.3 years). The visual analogue scale (VAS) for global rhinitis symptoms, nasal provocation test (NPT), anterior rhinomanometry, and T&T olfactometry were used to assess the short-term outcomes, preoperatively and postoperatively at the end of three months after surgical procedure. SPSS19.0 software was applied for statistical analysis., Results: At three months after treatment, the total nasal symptom VAS scores significantly decreased from 7.0 ± 2.0 to 2.5 ± 1.5 (X(-) ± s; t = 18.00, P = 0.0001). All patients were allergic to house dust mites with positive NPT before treatment. At three months from the coblation intervention, 88.0% of the patients changed from positive NPT to negative, while 12.0% remained as positive. There was a significant reduction in total nasal resistance, which diminished from 0.772 ± 0.224 to 0.221 ± 0.112 kPa·s·L(-1) after treatment (t = 22.00, P = 0.0001). Preoperative olfactory tests showed hyposmia in 31.0% of the patients, with 22 cases for slight and 9 cases for moderate disorder. Three months after treatment, 13.0% were diagnosed as hyposmia, with 7 cases for slight and 6 cases for moderate disorder (χ(2) = 10.44, P = 0.005)., Conclusion: Nasal coblation plasma surgery provides favorable short-term outcomes in terms of remarkable improvement in nasal symptoms, hyperreactivity of nasal mucosa, nasal flow and olfactory function in patients with moderate to severe PAR, but long-term effect needed further observation.

Objective: To investigate the effect of transfusion of apoptotic and necrotic thymocytes prior to sepsis on the survival rate of mice., Methods: BALB/c mice are divided into 3 groups and received intravenous injection of PBS (control), apoptotic thymocytes, or necrotic thymocytes. Three days later, cecal ligation and puncture (CLP) were performed to induce sepsis in these mice, and their survival and organ damage were observed., Results: The survival rates of mice in PBS group was 44.6% at the end of first week after CLP, and obvious lung and kidney damages were observed. A significant increase in the survival rate was found in apoptotic cell transfusion group (69.6%, P=0.012), with also lessened lung and kidney damages. The survival rate of mice in necrotic cell transfusion group was only 31.6% at 2 weeks, significantly lower than that in PBS group (P=0.035), and the lung and kidney damage was even more obvious., Conclusion: Transfusion of apoptotic thymocytes 3 days before induction of sepsis can reduce organ damage and improve the survival rate of mice, while necrotic cell transfusion produces the opposite effect.

This study was aimed to construct an adenovirus hybrid system with high transduction efficiency and site-specific integration. By a series of DNA manipulation, a hybrid system of two adenovirus vectors was constructed. One vector contains loxP-flanked transgene expression cassette, in which there are hFIX and DsRed coding sequences and attB for phiC31 recolonization. The other vector carries Cre and phiC31 gene. Vectors only expressing Cre or phiC31 were used as controls. 293A cells were constructed and transfected with the adenoviral vectors by Lipofectamine 2000, and the expression of target genes was identified by fluorescence microscopy and RT-PCR. The results showed that after being identified by PCR, restriction analysis and sequencing, an adeno-integrase hybrid system was successfully constructed. The system expressed RFP, GFP, hFIX, Cre and phiC31 in 293A cells in vitro. It is concluded that the adeno-integrase hybrid system is successfully constructed, which lays a good foundation for further investigation of its therapeutic application.

Objective: To investigate whether the plasmid bearing attB and human coagulation factor IX (hFIX) coding sequence could insert into hemophilia B mice genome and persistently express hFIX with co-injected integrase., Methods: The plasmid attB-hFIX-pIRES2-EGFP was constructed, which bore attB site and hFIX coding sequence and was proved in vitro to express hFIX. The plasmid and CMV-int expressing integrase was co-infused rapidly in a large-volume solution through tail vein of hemophilia B mice. Mice infused with the plasmid alone served as controls. ELISA was performed to determine serum hFIX level. Correction of coagulation defect in vivo by plasmid infusion was assessed by bleeding time. Genomic integration of the plasmid was determined by nested PCR., Results: The plasmid attB-hFIX-pIRES2-EGFP was successfully constructed. The hemophilia B mice produced (1533 ± 239) ng/ml hFIX at 24 hour after infusion of the hFIX encoding plasmid and the bleeding diathesis of the hemophilia B mice was significantly corrected as measured by clotting assays. However, whether or not co-injected with CMV-int, the serum hFIX level decreased to background level in 10 days after infusion. Nested-PCR results indicated that the integrase phiC31 resulted in the integration of the plasmid in the mouse liver chromosomes., Conclusion: Integrase phiC31 can catalyze recombination of 34 bp attB and pseudo-attP. Human FIX driven by CMV promoter can be transiently and highly expressed after infusion, but rapidly silenced in vivo.

Objective: To compare the responses to sepsis between C57BL/6 and BALB/c mice., Methods: Thirty C57BL/6 mice and 30 BALB/c mice were randomized into sham-operated group and sepsis group (n=15). Sepsis model was established by cecal ligation puncture (CLP) in the mice, and 6 h after the operation, 5 mice from each group were selected randomly for cytokine detection including IL-1beta, IL-2, IL-4, IL-5, IL-10, GM-CSF, IFN-gamma and TNF-alpha by Bio-plex. The other 10 mice in each group were used for survival analysis., Results: The survival rates of BALB/c and C57BL/6 mice were both 100% in one week after the sham operation, but lowered to 10% and 50% in one week after CLP, respectively. The survival rate of C57BL/6 mice was significantly lower than that of BALB/c mice (P<0.05). After CLP, C57BL/6 mice showed significantly greater IL-4, TNF-alpha and IL-10 production than the sham-operated mice, but the concentrations of the 8 cytokines in BALB/c mice after CLP showed no significant increment., Conclusion: Compared with BALB/c mice, C57BL/6 strain mouse is more sensitive to sepsis.

Combined deficiency of factor V and VIII (F5F8D) is a rare, autosomal recessive disorder caused by mutations of either lman1 or mcfd2. To identify mutations of these two genes in a Chinese F5F8D family, the samples of peripheral blood were collected from the proband and her parents. Coagulation tests were carried out, including activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT), fibrinogen (Fg) and coagulate activity of FV, FVIII (FV:C, FVIII:C). The genomic DNA was extracted, then all the exons and intron/exon boundaries of these two genes were amplified by polymerase chain reaction (PCR). The products were finally analyzed by direct sequencing. The results showed that the proband's APTT, PT, TT, Fg, FV:C and FVIII:C were 82.2 sec, 19.6 sec, 18.6 sec, 2.9 g/L, 7.1% and 18.7% respectively, while those parameters of the parents were all within the normal range. Two pathogenic mutations were identified in lman1 gene of the proband: one was the heterozygous c.912&#95;913insA in exon 8 resulting in a frameshift of p.Glu305fsX20; the other was the heterozygous c.1366C > T in exon 11 resulting in p.Arg456X. The proband's father and mother were heterozygous for c.1366C > T and c.912&#95;913insA respectively. It is concluded that F5F8D of the proband is caused by a novel compound heterozygous mutation of the lman1 gene, which has never been reported.

In order to evaluate whether mesenchymal stem cells (MSCs) from non-hematopoietic tissues are able to regulate megakaryocytopoiesis, we identified human MSCs from adult bone marrow (ABM), fetal pancreas (FPan) and umbilical cord (UC), and their abilities to support megakaryocyte (MK) differentiation from CD34(+) hematopoietic progenitor cells (HPCs) were comparatively studied. First, MSCs were isolated from ABM, FPan and UC then their growth kinetics, molecular characterization and mesodermal differentiation capacity were determined. ABM-MSCs, FPan-MSCs and UC-MSCs were irradiated and cocultured with human umbilical cord blood (UCB) CD34(+) cells, and the expansion efficiency of MK progenitor cells and MK formation were analysed and compared. Finally, SCF, IL-6 and GM-CSF expression by the three types of MSCs were also examined. Our results showed that FPan-MSCs and UC-MSCs shared most of the characteristic of ABM-MSCs, including morphology, immunophenotype, adipogenic and osteogenic differentiation potentials. Compared with ABM-MSCs, fetal MSCs had higher proliferative capacity. After 7 days' coculture, the maximal production of CD34(+)/CD41a(+) cells was obtained in a group of CD34(+) HPCs + ABM-MSCs. Furthermore, this group produced more MK colonies than other groups (p < 0.05). Surface antigen and ploidy analysis morphological observation demonstrated that a proportion of expanded cells in each group differentiated into mature MKs. ABM-MSCs, FPan-MSCs and UC-MSCs were revealed to express SCF, IL-6 and GM-CSF at mRNA level. We conclude that FPan-MSCs and UC-MSCs have the ability to promote megakaryocytopoiesis, while ABM-MSCs expand more MK progenitor cells from CD34(+) HPCs than MSCs from non-hematopoietic tissues and CD34(+) cells alone.

The present study was aimed to isolate and identify human mesenchymal stem cells from adult bone marrow (BM-MSC) and umbilical cord (UC-MSC), and to compare their ability to support in vitro long-term hematopoiesis. MSC from bone marrow and umbilical cord were isolated by using density gradient centrifugation or enzyme digestion. MSC were further purified by adherent culture. Immunophenotype, adipogenic and osteogenic differentiation potential of BM-MSC and UC-MSC were detected. The hematopoietic supporting capacity of BM-MSC and UC-MSC was assessed by LTC-IC assay. Nonadherent cells in each group were collected for phenotypic analysis at 3, 5 and 7th week of culture. The results showed that BM-MSC and UC-MSC in culture shared a similar spindle-shaped morphology and adhered to the tissue culture substrate. They were both positive for CD90, CD105, CD73, CD29, CD54, CD166, HLA-ABC, and negative for HLA-DR, CD34 and CD45. BM-MSC and UC-MSC could differentiate into adipocytes or osteoblasts confirmed by oil red O staining and von Kossa staining, separately. LTC-IC assay showed that at 5th week of culture, the difference of the CFC yields between UC-MSC group and BM-MSC group was not statistically significant (p>0.05). At 6, 7, 9th week of culture, the CFC yields in the UC-MSC group were lower than those of BM-MSC (p<0.05). The phenotypic analysis of nonadherent cells at 3, 5, 7th week of culture indicated that along with prolongation of time, the percentages of CD34+ cells and CD117+ cells in each group decreased markedly, and the percentages of CD33+ cells, CD13+ cells and CD11b+ cells increased gradually. It is concluded that MSC from human adult bone marrow and umbilical cord can be successfully isolated and identified. UC-MSC are able to support long-term hematopoiesis in vitro, but its hematopoietic supportive capacity is weaker than those of BM-MSC.

Inherited afibrinogenemia is a rare autosomal recessive bleeding disease characterized by complete absence of fibrinogen in blood. To identify the genotype in a Chinese family with inherited afibrinogenemia, the samples of peripheral blood were collected from 6 members of 3 generations. The activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT) and fibrinogen (Fg, clauss) were tested. Fg was also analyzed by using immunoturbidimetry method. DNAs of six members were extracted by using a DNA extract kit. All the exons and exon-intron boundaries of the three fibrinogen genes were amplified by using PCR and analyzed by direct sequencing. The results showed that the parents of proband were 3 degree consanguinity. A homozygous c.934&#95;935insA in FGA was found in proband which results in the change of protein p.Ser312fsX42. The parents, grandmother, maternal grandmother and father's sister were all detected with heterozygous mutation which was same as that in proband. In conclusion homozygous c.934&#95;935insA in FGA is a cause of inherited afibrinogenemia and a novel mutation being reported.

The present study was purposed to evaluate the safety of mesenchymal stem cell (MSC)-based therapy impacting on atherosclerosis. Allogeneic MSCs were obtained from rabbit bone marrow aspirates and expanded in vitro. New Zealand white rabbits were divided into three groups: 24 rabbits with hypercholesterolemia receiving intravenous injection of either 5 x 10(7) MSCs (n = 12) or saline (n = 12) after 5 weeks on a high lipid diet and additional rabbits (n = 6) fed with standard rabbit diet were served as controls. Body weight and blood lipids were measured at weeks 0, 5, 9 and 13 during the study. All rabbits were sacrificed at week 13. Atherosclerotic lesion size and vasa vasorum were evaluated by using pathological analysis and immunocytochemical technique. The results showed that the aortic sinus lesion size significantly increased in rabbits infused with MSCs as compared with controls receiving saline (23.35 +/- 3.51% and 11.39 +/- 3.08% respectively). The lesion size in whole aortas of MSC-treated rabbits was 76.64 +/- 12.70% versus 57.61 +/- 9.00% in saline-treated animals (p < 0.05). Moreover, vasa vasorum networks in MSC-treated aortas were more numerous and had increased capillary density. It is concluded that the allogeneic MSC transfusion may result in an increase in atherosclerotic lesion size. In cell therapy with MSCs or cell populations containing MSCs a strategy to attenuate the high potential of MSCs involved in atherogenesis of atherosclerosis should be taken in account.

This study was aimed to design and screen short hairpin RNA (shRNA) molecules targeting multidrug resistance gene (mdr1), as well as to investigate the effects of shRNA expression vector on K562/A02 cells. Mdr1-shRNA expression vector was transfected into K562/A02 cells by lipofectamine 2000, and G418 was added to screen and establish the stable expression cell strain. The expressions of mdr1 mRNA and protein were detected by real-time RT-PCR and Western blot respectively. The sensitivity of cells to chemodrugs after interference were tested by CCK8 assay. The function of p-glycoprotein was determined by Rhodamine 123 efflux experiment. The results showed that all of 4 mdr1-shRNA expression vectors could significantly knockdown the expression of p-glycoprotein as compared with control vector, moreover, the vector targeting 508 - 526 sites of mdr1 gene was the best one. It is concluded that the mdr1-shRNA expression vector gained by screening can significantly knockdown the expression of mdr1 gene and reverse leukemia drug resistance, paving the way for the application of RNAi in the following animal experiments.

Objective: To determine whether mobilized peripheral blood mononuclear cells (M-PBMNCs) obtained from patients with diabetes was impaired in therapeutic neovascularization in limb ischemia, and to explore the pathological mechanisms of the impairment., Methods: Endothelial progenitor cells (EPC) were cultured in EGM-2MV, and then characterized by uptake of 1, 1-dioctadecyl-3, 3, 3, 3-tetramethylindocarbocyanine-labeled acetylated low density lipoprotein (Dil-AcLDL) and binding of ulex europaeus agglutinin (UEA). The number of EPC was compared between M-PBMNCs obtained from diabetic patients and those from normal subjects. M-PBMNCs obtained from diabetic patients, M-PBMNCs obtained from normal controls, or PBS were injected into the ischemic limbs of streptozotocin-induced diabetic nude mice. The limb blood perfusion was detected by laser Doppler blood perfusion imaging between these three groups in the following 1, 3, 7, 14, 21, and 28 days. Ambulatory score and ischemia damage were evaluated in the following 4 weeks. Capillary/fiber ratio was detected by CD31 or BS-1 lectin, and arteriole density was detected by alpha-smooth muscle actin (alpha-SMactin)., Results: The number of EPC from diabetic patients were positively correlated with the blood perfusion (R = 0.486, P < 0.05) and capillary density (R = 0.491, P < 0.05), and the EPC number in diabetic patient were negatively correlation with their disease courses (R = - 0.587, P < 0.05). Transplantation of diabetic M-PBMNCs augmented the blood perfusion of ischemia hindlimbs, increased the capillary and arteriole densities, and promoted the collateral vessel formation. However, all the improvements were less significant in the diabetic patients than in the non-diabetic patients (P < 0.05)., Conclusion: Diabetes decreased the capability of M-PBMNCs to augment neovascularization in ischemia.

Objective: To explore the mechanism of the therapeutic efficiency of mobilized peripheral blood mononuclear cells (PBMNCs) with and without CD34+ cell depletion in ischemia nude mice., Methods: After femoral ligation of mice, 1 x 10(6) PBMNCs, CD34+ cell depletion PBMNCs, or PBS were intramuscularly injected into the ischemic limb. Blood perfusion, ischemia damage, and capillary density of the limb were observed. VEGF expression in ischemic limbs was assayed by ELISA and immunohistochemistry., Results: PBMNCs transplant greatly improved the recovery of ischemic limbs. At day 28 after surgery, the blood perfusion rate of ischemic limbs recovered to (96.4 +/- 5.6)% from (20.3 +/- 4.2)% in PBMNCs transplanted group, compared with (71.3 +/- 4.4) % in PBS group (P <0.01). Depletion of CD34+ cells reduced the perfusion ratio to (83.8 +/- 5.2)% (P < 0.05). Capillary density in PBMNCs transplanted group was (521 +/- 47)/mm2, while in CD34+ cell-depleted group [ (396 +/- 21)/mm2] (P < 0.05). PBMNCs were found to incorporate into vascular network. VEGF was greatly up-regulated after transplantation of PBMNCs and was secreted in situ., Conclusion: Transplantation of mobilized PBMNCs augments neovascularization in ischemic limb via supply of stem/progenitor cells and angiogenic factors. Depletion of CD34+ cells impaired therapeutic efficacy for limb ischemia.