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AIM: To investigate the effect of FAK - related non - kinase ( FRNK) on the expression of membrane - type matrix metalloproteinase -1 ( MT1 - MMP) in hepatic stellate cells ( HSC). METHODS:FRNK were trans-fected into HSCs by cationic liposome method. The protein levels of FRNK in HSC were assayed by Western blotting. The levels of MT1 - MMP were determined by RT - PCR for mRNA and by Western blotting for protein, respectively. RESULTS: The up -regulated expression of FRNK protein was observed and it was at 48 h after transfection that the FRNK protein content was the highest ( P < 0.05 ). The expressions of MT1 - MMP mRNA and protein were also up - regulated by the transfection of FRNK, and it was at 48 h after transfection that the MT1 - MMP protein content was significantly increased. CONCLUSION: The mRNA and protein of FRNK were over - expressed in HSC transfected with the gene of FRNK. The inhibitory effect of FRNK on the collagen synthesis in HSC may be through the up - regulation of MT1 - MMP.
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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.
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RNA interference(RNAi),the first method of choice to suppress gene expression in vitro,is a conserved biologic response to double-stranded RNA that resulted in the sequence-specific silencing of target gene expression.Recently,more than 100 literatures reported synthetic small interfering RNAs in mammals.These reports demonstrated that RNAi is also a powerful tool for in vivo research and may become an effective therapeutic tool.This paper reviews the influencing factors and experimental design of in vivo use of RNAi.
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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