The Impact Of Cholesterol On The Bile Duct Fibroblasts Proliferation And Phenotypic Transformation

The disorder of cholesterol metabolism is an important cause of biliary diseases. Previous studies suggest that cholesterol (Ch) can change the motility of cholecyst, the function of gallbladder contraction decreases in patients with cholesterol calculus and in animals with hypercholesterolemia, which could lead to formation of cholesterol calculus.Researchers consider that cholesterol metabolism malfunction has an effect on the structure and function of bile duct and sphincter of bile duct (SBD). We found that cholesterol liposome (CL) affected not only the construction and quantity of cytoskeleton but also proliferation of rabbit SBD smooth muscle cells.There are many fibroblasts in biliary system except that SBD is formed predominantly with smooth muscle cells, but it is still unknown if Ch has any effect on bile duct fibroblasts and fibroblasts play an important role in the configuration variation of bile duct. We studied the effect of Ch on the proliferation and phenotype of fibroblasts and its mechanism on the basis of bile duct cell culture to find out the effect of fibroblast in bile duct remodeling.1. Aims(1) To investigate how cholesterol can affect the proliferation of bile duct fibroblasts, and to probe its possible mechanism.(2) To investigate the effects of cholesterol on the phenotype of bile duct fibroblasts of rabbits.2. Methods(1) The cultured bile duct fibroblasts were divided randomly into three groups: control group, middle concentration group, high concentration group.(2) The growth of bile duct fibroblasts was measured respectively bythe means of MTT.(3) Cell cycle of bile duct fibroblasts was examined respectively by the means of flow cytometer (FCM).(4) The effect of cholesterol on the intracellular level of PCNA was measured by the means of immunohistochemistry stain respectively.(5) 16 rabbits were divided randomly into two groups: the control group and the experiment group. The rabbits in experiment group were fed with hypercholesterol diet for 8 weeks.(6) Bile duct was dissociated from rabbits and prepared for transmission electron microscopy.(7) The purified bile duct fibroblasts were cultured and divided randomly into three groups: control group, cholesterol middle concentration group (0.6g/L), cholesterol high concentration group (1.2g/L).After incubated for 72h, the fibroblasts were made into specimens for transmission electron microscopy.(8) The expression of a-actin hi bile duct fibroblasts was measured by means of laser scanning confocal microscopy.3. Results(1) Middle concentration group (0.4-0.8g/L) significantly increased the optical absorbance value at 490 nm (A490nm) of bile duct fibroblasts. The value (A490nm) of control group was 0.249+0.025, and Ch (0.6g/L) increased it to the maximum (0.486+0.019, P<0.01 vs control group), while Ch (1.2g/L) decreased it to 0.163+0.018 (P<0.01 vs control group).(2) Flow cytometric analysis indicated that middle concentration group (0.6g/L) increased the number of bile duct fibroblasts in S-phase (from 11.7% to 33.4%).(3) There was a high expression of PCNA in bile duct fibroblasts of middle concentration group (0.6g/L), the colouration intensity ofmiddle concentration group (126.3+10.2%AU) was significantly higher than that of control group (88.3+11.6%AU). High concentration group (1.2g/L) decreased the expression of PCNA, the colouration intensity was 96.3+11.4% AU (P<0.05 vs middle concentration group).(4) With the transmission electron microscopy, the normal bile duct fibroblasts were shuttle-shaped, and there were abundant rough endoplasmic reticulums (RER), but few mitochondria or microfilaments in cytoplasm. This is the typical phenotype of fibroblasts. Bile duct fibroblasts of hypercholesterolemic rabbits were observed by the transmission electron microscopy. Rough endoplasmic reticulums in fibroblasts were significantly reduced, with a lot of microfilament bundles or stress fibers appea...