Effects Of Bile Salt On Cell Proliferation In Human Bile Duct Fibroblasts

Objective:Bile salt play an important role in the biliary tract physiological, and the bile salt overload can lead to cell apoptosis and necrosis. However,studies of the efect of the low-concentration bile salt on bile duct fibroblasts proliferation are still few. To investigate the efect of the low-concentration bile salt on bile duct fibroblasts proliferation and TGF-β1、Smad3、α-SMA protein expression. It provides us a theoretical foundation for clinical preventation and treatment of human benign bile duct scar.Methods:Human bile duct scar-derived fibroblasts(HBSFs) and normal fibroblasts(HBNFs) were cultured in primary culture by trypsin digestion combined with tissue block method, then they were incubated in medium containing0、0.625、1.25、2.5、5、10、20、40、80umol/L bile salt for24、48、72hours. The cell survival rate was determined by MTT assay. And then to determine the optimum concentration of bile salt for promoting proliferation of human bile duct fibroblasts. After Human bile duct fibroblasts were stimulated with bile salt in optimum concentration for24hours.The cell cycles were detected by flow cytometry. and the relative percentage of S phase was calculated. The expression of TGF-β1、Smad3and a-SMA protein was detected by immunocytochemistry and western blotting.Results:1. HBSFs and HBNFs cultured in vitro were all spindle-shaped, vimentin-positive and keratin-negative, which characterize them as fibroblast.2. The proliferation of the HBNFs which were stimulated with low-concentration (0.625-10.0umol/Lumol/L)bile salt were higher than the control group, and difference was statistically significant(P<0.05). And as the extension of time, the role of promotion enhanced(P<0.05).Moreover, the relative percentage of S phase were significantly increased by the optimum concentration1.25umol/L) of bile salt(P<0.05).3. Both TGF-β1and Smad3expressed in cytoplasm of HBSFs and HBNFs. Their expression were higher in HBSFs than HBNFs. a-SMA almost no expression in HBNFs. but in the HBSFs has a higher level of expression.4. After stimulated with low-concentration bile salt, the expression of TGF-β1、Smad3and a-SMA increased significantly in HBNFs, and nuclear translocation of Smad3were found.Conclusions:Low-concentration bile salt can promote proliferation of human benign bile duct normal fibroblasts, and it is time-dependent. Bile salt can also induce phenotype modulation of the cultured fibroblasts, namely transforming to MFB-like cells. And which might be associated with TGF-beta1/Smad3pathways.