[Effects of exogenous TGF-β3 on the expression of endogenous TGF-β3 in hepatic stellate cell-T6 (HSC-T6)].

Objective To investigate the effects of exogenous TGF-β3 on the expression of endogenous TGF-β3 in hepatic stellate cell (HSC).Methods HSCs were cultured and divided into two groups:TGF-β3group and blank control group,the cells of TGF-β3 group were exposed to TGF-β3 (10 ng/ml),whereas the blank control group was not treated.The cells were incubated in the presence of exogenous TGF-β3 and then (1)were harvested at 0h,lh,2h,4h,12h,24h,and real time PCR was performed to detect the mRNA expression of endogenous TGF-β3.(2) The cells were collected at 0h,lh,6h,12h,and western-blot was used to detect the protein synthesis of endogenous TGF-β3 in HSC; (3) The cell culture supematant was harvested at 0h,lh,2h,4h,8h,14h,24h,and ELISA was performed to measure the total protein of extracellular TGF-β3; HSCs were treated with TGF-β3 (10ng/ml) for 2h.The cells were then incubated in serum-free medium and the cell culture supernatant was harvested at 2.25h,2.5h,3h,4h,6h,10h and 14h.ELISA was used to detect the extracellular secret ion of endogenous TGF-β3 by HSCs.Results (1) Exogenous TGF-β3 treatment induced a marked increase in TGF-β3 mRNA expression.By 2h of exogenous TGF-β3 treatment,maximal TGF-β3 mRNA expression levels (2.796 ± 0.518) of 2.74 fold above control values (1.022 ± 0.038) was reached (P ＜0.05).Thereafter,TGF-β3 mRNA expression level declined,and the expression level was maintained at level of 1.45-fold for at least 10h and was 1.18-fold above control values by 24h TGF-β3 treatment (P ＜ 0.05); (2)No significant difference about the intracellular protein expression level of endogenous TGF-β3 was found between two groups.(P ＞ 0.05); (3) The total expression level of TGF-β3 reached a peak [(18.931 ± 2.904)ng/ml] at 4h after TGF-β3 treatment (1.89-fold higher than basic TGF-β3 (10ng/ml).After that,it slowly declined.The expression peak [(0.835 ± 0.027) ng/ml] induction of extracellular secreted TGF-β3 was at 3h (32.12-fold higher than control [(0.026 ± 0.022) ng/ml],(P ＜ 0.05).Thereafter,TGF-β3 slowly decreased after the peak time,and their expressions were still statistically significant as compared to the control (P ＜0.05).Conclusion Exogenous TGF-β3 could increase the expression of endogenous TGF-β3 mRNA and extracellular secreted TGF-β3 protein obviously. Key words: Liver cirrhosis;  Hepatic stellate cell;  Transforming growth factor beta