A Study On The Effect Of Bile On Human Fibroblasts In Vitro Culture

Objective To study the effect of bile on the cultured human fibroblasts so as to explore the role of bile in the formation of biliary scars and relative mechanisms. Methods Human bile was collected in the operations of the patients with polypoid lesion of gallbladder. After being percolated, different concentrations of bile was added into the culture medium. Morphological changes in both control group and trial group were observed under inverted microscope and electron microscope. The effect of different concentrations of bile on the proliferation of cultured fibroblasts was studied by MTT assay. And the apoptosis rate and cell circle distribution were determined by FCM.Results Inverted microscopy showed that fibroblasts in trial group had a larger quantity and a higher density compared with the control group. The HE staining result showed that cells in control group had large nucleuses and small nucleoluses. While cells in trial group had large nucleuses and large nucleoluses. Pieces of lightly stained red material could also be seen among the cells in trial group. Electron microscopy showed that fibroblasts in control group had large nucleuses and abundant RER. But few microfilaments was observed in the cells in control group. And apoptic cells could be seen in control group. While cells in trial group had large nucleuses and large nucleoluses. More abundant RER and Golgi complex could be observed. Some expanded RER could also be seen. A large amount of microfilament bundles was observed in the cells. And collagen-like material could also be seen among the cells in trial group. Forty eight hours after bile of 20-100 times dilution were added into the culture medium respectively, the result of MTT assay showed that the correspondent OD values became larger when the concentrations of bile became higher. There were statistically significant differences between the control group and the trial group in the effect of bile at different concentrations on the proliferation of fibroblasts in vitro culture. And the effect was concentration-dependant to some extent. FCM results showed that apoptosis rates of cells in 100 times dilution group and 50 times dilution group were 11.61±2.83% and 9.62±2.45% respectively. While the value of the control group was 20.11±2.61%. There were statistically significant differences between the control group and the trial group. And cell circle distribution results showed that the proportion of cells in G0/G1 stage in 100 times dilution group and 50 times dilution group were 54.2±4.2% and 49.8±5.9% respectively. While the value of the control group was 71.1±10.0%. There were statistically significant differences between the control group and the trial group. Conclusion Human Bile enhances proliferation of cultured human fibroblasts and inhibits their apoptosis. And Bile can promote the entry of cultured fibroblasts into S phase from G0/G1 phase. Bile can also induce phenotype modulation of the cultured fibroblasts, namely transforming to MFB-like cells. Hence bile can promote the formation of biliary scars. Biliary mucous membrane plays an important role in preventing the proliferation of fibroblasts induced by bile.