Objective To study the presence of CD4+CD25+regulatory T cells and hepatitis B virus(HBV)specific cytotoxie T lymphocyte(CTL)in peripheral blood and liver tissues of patients with chronic hepititis B(CHB)and its clincial significance.Methods One hundred and fifty-seven HBV-infected patients,including 20 cases of acute hepatitis B,115 cases of chronic hepatitis B,and 22cases of HBV-related liver cirrhosis,and 20 healthy controls were enrolled in this study.Peripheral blood was collected and liver tissues were obtained from some of the enrolled subjects.The CD4+CD25+regulatory T cells and HBV specific CTL were analyzed using flow cytometry and cytokine flow cytometry(CFC).The comparison between groups was done by t test.Results The percentages of CD4+CD25+ regulatory T cells in the peripheral blood of patients with acute hepatitis B and CHB of mild,moderate and severe degree were(2.87±0.94)%,(3.53±1.56)%,(4.59±2.98)%and(3.65±1.73)%,respectively,which were higher than that of controls(2.36±0.60)%(t=2.04,5.97,3.30 and 3.17,respectively,P<0.01).The percentages of HBV specific CTL in the peripheral blood of patients with mild,moderate and severe degree of CHB and HBV-related liver cirrhosis were (0.189±0.152)%,(0.103±0.110)%,(0.118±0.120)%and(0.098±0.101)%,respectively,which were significantly lower than that of acute hepatitis patients [(0.815±0.360)%](t=10.09,11.87,9.17 and 8.96,respectively,P<0.01).CD4+CD25+ regulatory T cells and HBV specific CTL in liver tissues were both higher than those in the peripheral blood.Conclusion CD4+CD25+regulatory T cells may play an important role in anti-HBV immune response through inhibiting CD8+T eell function.

Objective To investigate the down regulation of phosphatase and tensin homolog deleted on chromosome 10 ( PTEN) gene by adenovirus mediated short hairpin RNA ( shRNA) on the adhesion in activated hepatic stellate cells( HSC ) in vitro and the related signal transduction mechanism .Methods The recombinant adenovirus ( Ad-shRNA/PTEN) with shRNA targeting PTEN and expressing green fluorescent protein ( GFP) were transient trans-fected into the cultural activated HSC in vitro.The experimental group as follows:1)Control group, viral medium was replaced by DMEM at virus transfection step .2 ) Ad-GFP group , HSC were infected with adenovirus expressing GFP alone.3)Ad-shRNA/PTEN group, HSC were infected with adenovirus both taking shRNA targeting PTEN and expressing GFP.PTEN mRNA expression was detected by RT-qPCR, and western blot was used for detecting pro-tein expressions of PTEN , focal adhesion kinase ( FAK) and phosphorylated FAK ( Thr397 ) [ p-FAK( Tyr397 ) ] in HSC.The toluidine blue stain method and MTT colorimetric method were used to determine the adhesion ability of HSC.Results When HSC were infected by adenovirus for 48 hours, PTEN protein and mRNA expressions in Ad-shRNA/PTEN group significantly decreased ( P<0.05 ) , compared to control group and Ad-GFP group, and the expressions of p-FAK ( Tyr397 ) in Ad-shRNA/PTEN group were significantly higher than those in control group and Ad-GFP group ( P<0.05 ) .The adhesion cell counting and the adhesion rate of HSC in Ad-shRNA/PTEN group significantly increased as compared with control group and Ad-GFP group ( P<0.05 ) .Conclusions The down-regulation of PTEN expression can promote the adhesion by increasing the activation of FAK signaling trans -duction in activated HSC in vitro.

Objective To investigate the effects of over-expression of wild-type PTEN gene and its mutant (G129E) on apoptosis and proliferation of activated hepatic stellate cells (HSC) in vitro and its potential mechanisms. Methods The activated HSC cells were cultured in vitro and transfected with PTEN gene and G129E gene via adenoviral vector. The apoptosis of HSC was measured by MTT , and its proliferation was assessed by TUNEL and flow cytometry (FCM) . Western blotting and real-time fluorescent quantitation PCR were used to detect expression of PTEN in HSC. And the changes of Bcl-2 and Bax expression were tested by Western blotting. Results The wild type PTEN gene and G129E gene were successfully transducted into HSC, which resuted in elevated expression of Bax and reduced expression of Bcl-2 (P<0.01). After transduction, HSC proliferation was markedly inhibited with inhibitory rates of 14.03% and 23.12% at 48 and 72 hours in Ad-PTEN ,respectively, as well as 9.52% and 12.63% in Ad-G129E, respectively. Apoptotic rate of HSC exposed to Ad-PTEN or Ad-G129E for 72 hours increased significantly (P<0.01). Furthermore, wild type PTEN was more powerful than G129E for above-mentioned effects. Conclusions Over-expression of wild type PTEN and its mutant G129E can inhibit the proliferation of activated HSC, and induce HSC apoptosis through the Bcl-2/Bax pathway. In addition, the effect of wild type PTEN is more powerful than that of G129E.

Objective Using an adenoviral vector , the wild-type PTEN gene was transduced into activated hepatic stellate cell (HSC) in vitro and the phosphorylation status of Akt were investigated. Methods The wild type PTEN gene was transduced into activated HSC in vitro mediated by adenoviral vector. The expressions of PTEN and total Akt in HSC were measured by Western blot and Real-time fluorescent quantitation PCR. And the expressions of phosphorylated Akt (Thr308) in HSC was determined by Western blot. Results The data showed that exogenous wild type PTEN gene was successfully transduced and expressed in activated HSC in vitro. The over-expression of wild type PTEN resulted in the significant down-regulated expression of phosphorylated Akt (Thr308) in activated HSC (P < 0.01). But no significant defferences were found in the expression of total Akt in activated HSC at both transcriptional and translational levels(P>0.50). Conclusions The overexpression of wild-type PTEN can negatively regulate PI3K/Akt signaling transduction by inhibiting the phosphorylation of Akt in activated HSC in vitro.

Objective To explore the relationship of phosphatase and tensin homology (PTEN) deleted on chromosome ten with the apoptosis of hepatic stellate cells (HSCs) in liver tissues of rats with hepatic fibrosis induced by bile stagnation.Methods Fifty adult male SD rats were randomly divided into model group (n=40) and sham operation group (n=10).The model of hepatic fibrosis was reproduced in model group by common bile duct ligation (BDL).Liver tissue of rats in model group (1,2,3 and 4 weeks after BDL) and sham operation group were obtained.PTEN expression in liver tissue was detected by immunohistochemistry.Apoptosis of HSC was determined by dual staining of terminal deoxynucleotidy transferase UTP-nick end labeling (TUNEL) and ?-SMA immunohistochemistry.Results Only a few apoptotic HSCs were found in normal livers.With the development of liver fibrosis,the expression of PTEN decreased gradually (P

Objective:To investigate the dynamic expression of protein tyrosine phosphatase SHP2 in liver tissue of rats with carbon tetrachloride (CCl 4)-induced liver fibrosis. Methods:Rat liver fibrosis model was established by intraperitoneal injection of CCl 4. Rat liver tissue histopathological changes were detected by HE and Masson-trichrome staining. Immunohistochemical staining, Western blot and real-time fluorescent quantitative PCR were used to detect SHP2 protein and mRNA expression in rat liver tissue. One-way analysis of variance was used for the comparison of means between multiple groups, and the LSD test was used for further inter-group comparison. Results:CCl 4-induced rat liver fibrosis model was successfully constructed, and with the extension of modeling time, the degree of liver fibrosis in rats were aggravated gradually. Immunohistochemical staining results showed that SHP2 was mainly expressed in the cytoplasm of rat liver tissues. With the aggravation of liver fibrosis, the number of cells with positive expression of SHP2 was aggravated gradually ( P < 0.05). Western blot and real-time fluorescent quantitative PCR results showed that the expressions of SHP2 protein and mRNA in rat fibrotic liver tissues at different times in week 2, 4, 6, and 8 were higher in modeling than control group ( P < 0.05), and was aggravated gradually with the liver fibrosis aggravation ( P < 0.05). Conclusion:The expression of SHP2 protein and mRNA in the liver tissue of rats with CCl 4-induced liver fibrosis increased gradually with the degree of liver fibrosis, and the degree of increase was consistent with the degree of liver fibrosis.

Objective:To investigate the effect of adenovirus-mediated short hairpin RNA (shRNA) downregulating SH2 domain-containing protein tyrosine phosphatase 2 (SHP2) on the apoptosis of human hepatic stellate cells LX-2 cultured in vitro.Methods:The recombinant adenovirus Ad-shRNA/SHP2 carrying shRNA targeted SHP2 and expressing green fluorescent protein (GFP), and the empty control virus Ad-GFP expressing GFP were transfected into LX-2 cells cultured in vitro. Real-time fluorescence quantitative PCR was used to detect SHP2 mRNA expression in LX-2 cells. Western blot was used to detect the protein expressions of SHP2, Bax, and Bcl-2 in LX-2 cells. TUNEL and annexin-V/propidium iodide dual-labeled flow cytometry were used to detect apoptosis in LX-2 cells. Experimental group: (1) Control group: LX-2 cells were transfected with DMEM instead of adenovirus; (2) Ad-GFP group: transfected with empty virus Ad-GFP; (3) Ad-shRNA/SHP2 group: transfected with recombinant adenovirus Ad-shRNA/SHP2. The means between multiple groups were compared using a one-way ANOVA and the LSD test was used for inter group comparisons.Results:shRNA-targeted SHP2 significantly down-regulated the expression of SHP2 protein and mRNA in LX-2 cells ( P < 0.05). The TUNEL and annexin-V/propidium iodide dual-labeled flow cytometry results showed that the apoptosis rate of LX-2 cells in the Ad-shRNA/SHP2 group (12.755%±1.606%, 19.340%±2.505%) ( P < 0.05) was significantly higher compared to the control group (3.077%±0.731%, 9.438%±0.804%) and the Ad-GFP group (3.250%±0.851%, 8.893%±1.982%), with no statistically significant difference between the control group and the Ad-GFP group ( P > 0.05). Western blot analysis of Bax and Bcl-2 protein expression in LX-2 cells of each group revealed that the Bax protein expression was significantly higher in the Ad shRNA/SHP2 group (2.493 ± 0.203) ( P < 0.05) compared to the control group and Ad-GFP group (1.989 ± 0.147, 1.999 ± 0.162), with no statistically significant difference between the control group and the Ad-GFP group ( P > 0.05), while the Bcl-2 protein was significantly decreased in the Ad-shRNA/SHP2 group (1.042±0.148) compared with the control group and the Ad-GFP group (1.707±0.146, 1.521±0.142), with no statistically significant difference between the control group and the Ad-GFP group ( P > 0.05). Conclusions:SHP2 expression down-regulation induces apoptosis of human hepatic stellate cells LX-2 in vitro by reducing Bcl-2/Bax.

Objective: To investigate the role of adenovirus-mediated short hairpin RNA (shRNA) in down-regulating the expression of phosphatase and tensin homologue deleted on chromosome ten (PTEN) on p130Crk-related substrates(p130Cas) and paxillin signal transduction to activate hepatic stellate cell (HSC) in vitro. Methods: The rat hepatic stellate cell line, HSC-T6 was cultured and activated in vitro. The adenovirus was used as a vector to transiently transfect shRNA targeting PTEN to activate HSC in vitro, and then PTEN low expression model of activated HSC in vitro was established. Western blot and real-time fluorescence quantitative PCR were used to detect the protein and mRNA expression of PTEN, p130cas and paxillin in activated HSC. The experiment was divided into control group (HSC were transfected with DMEM medium instead of adenovirus), Ad-GFP group (HSC were infected with empty the adenovirus expressing green fluorescent protein (GFP) alone), and Ad-shRNA/PTEN group (HSC were infected with the recombinant adenovirus containing both shRNA targeting PTEN and GFP gene). One-way analysis of variance was used for comparison of multiple groups, and LSD test was used for inter-group comparison. Results: shRNA targeting PTEN was successfully transfected and significantly down-regulated the PTEN protein and mRNA expression of HSC in vitro (P < 0.05), and the PTEN low expression model of HSC in vitro was successfully constructed. Compared with the expression of p130cas mRNA in the three groups of HSC, the expression fold of p130cas mRNA in the Ad-GFP group and the Ad-shRNA / PTEN group was 1.01 times and 1.52 times, respectively. The expression of p130cas mRNA in HSC of the Ad-shRNA / PTEN group was significantly higher than control group and Ad-GFP group (P < 0.05), but there was no statistically significant difference between the control group and the Ad-GFP group (P > 0.05). The expression of p130cas protein in the three groups was higher than that in the control group (0.74 ± 0.07) and the Ad-GFP group (0.72 ± 0.02); P < 0.05, but there was no statistically significant difference between the Ad-GFP group and the control group (P > 0.05). The expression of paxillin mRNA in the three groups of HSCs was compared with the expression of paxillin mRNA in the control group of HSC being 1, the expression folds of paxillin mRNA in the Ad-GFP group and Ad-shRNA / PTEN group were 0.97 times and 1.58 times, respectively. The expression of paxillin mRNA in the Ad-shRNA / PTEN group was higher than that in the control group and the Ad-GFP group (P < 0.05), and there was no statistically significant difference between the control group and the Ad-GFP group (P > 0.05). The expression of paxillin protein in the three groups of HSCs was higher in the Ad-shRNA / PTEN group (0.91 ± 0.05) than control group (0.46 ± 0.03) and Ad-GFP group (0.50 ± 0.04), P < 0.05, and there was no statistically significant difference between the Ad-GFP group and the control group (P > 0.05). Conclusion Down-regulation of PTEN expression can significantly boost p130cas and signal transduction activity of paxillin protein in activated HSC in vitro.

Objective: To investigate the effect of overexpression of wild-type phosphatase and tensin homolog (PTEN) deleted on chromosome 10 and its mutant G129E (exhibiting the activity of protein phosphatase and losing the activity of lipid phosphatase) on F-actin in activated hepatic stellate cells (HSCs) cultured in vitro. Methods: The activated hepatic stellate cell-T6 (HSC-T6) cells were cultured in vitro, and activated HSCs were transfected with adenovirus that carried wild-type PTEN gene and G129E gene using transient transfection. The HSCs were divided into the following groups: control group, which was transfected with DMEM medium instead of virus solution; Ad-GFP group, which was transfected with the empty adenovirus vector with the expression of green fluorescent protein (GFP); Ad-PTEN group, which was transfected with the recombinant adenovirus with wild-type PTEN gene and GFP expression; Ad-G129E group, which was transfected with the recombinant adenovirus with G129E gene and GFP expression. Western blot and quantitative real-time PCR were used to measure the protein and mRNA expression of PTEN in activated HSCs; under a laser scanning confocal microscope (LSCM), phalloidine labeled with the fluorescein tetramethylrhodamine isothiocyanate (TRITC) was used to observe the morphology of HSCs, distribution and fluorescence intensity of F-actin, and changes in pseudopodia and stress fibers, and a calcium fluorescence probe (Rhod-2/AM) was used to measure the changes in Ca²⁺ concentration in HSCs. A one-way analysis of variance was used for comparison between multiple groups, and the least significant difference test was used for comparison between two groups. Results: Wild-type PTEN and G129E genes were highly expressed in activated HSCs. In the control group and the Ad-GFP group, HSCs had a starlike or polygonal shape, F-actin was reconfigured and formed a large number of stress fibers which stretched across the whole cell, and layered pseudopodia were seen around the cell. In the Ad-PTEN group and the Ad-G129E group, the HSCs had a fusiform shape, F-actin was mainly seen around the cell, a small number of stress fibers were seen inside the cell, and layered pseudopodia around the cell disappeared. The Ad-PTEN group and the Ad-G129E group had significant reductions in the fluorescence intensity of F-actin compared with the control group and the Ad-GFP group (357.67±13.39/377.25±14.55 vs 961.87±27.33/954.68±20.71, F = 1783.486, P < 0.05), while there were no significant differences between the Ad-PTEN group and the Ad-G129E group, as well as between the control group and the Ad-GFP group (P > 0.05). The Ad-PTEN group and the Ad-G129E group had significant reductions in the relative concentration of Ca²⁺ compared with the control group and the Ad-GFP group (251.60±90.88/352.18±146.01 vs 1953.95±132.99/1937.57±115.17, F = 834.988, P < 0.05), while there were no significant differences between the Ad-PTEN group and the Ad-G129E group, as well as between the control group and the Ad-GFP group (P > 0.05). Conclusion The overexpressed wild-type PTEN and its mutant G129E can significantly inhibit the formation and reconfiguration of cytoskeletal protein F-actin and reduce the concentration of Ca2+ in activated HSCs in vitro. In addition, there are no significant differences in the above effects between wild-type PTEN and G129E.