| Objective:This thesis is divided into three parts, the first part, through2AAF+2/3PH standardization method to establish a rat model of hepatic oval cell activation and liver function test indicators, to determine liver cell damage, and can successfully activated hepatic oogonium and the purpose of the preliminary appraisal. The second part, through the rat hepatic oval cells cultivation and induction, and at the same time in the process of inducing cell surface marker detection and observation, to achieve successful induction of hepatic oval cells differentiation become the purpose of the bile duct epithelial cells. The third part, through the adoption of real-time fluorescence quantitative method to detect bile duct epithelial cells induced by hepatic oval cells type of micrornas in different differentiation periods of temporal expression level, discusses corresponding micrornas regulate the expression of target in different developmental period in the known differences. To study and explore each of micrornas in the whole process of bile duct epithelial cells regeneration to repair the purpose of the role and significance.Methods:The first part, the model of rat hepatic oval cell activation, set up the negative control model group, respectively collected20on rat hepatic oval cell activation and liver specimens were negative control group and control model group and blood specimens. For pathological inspection and TBIL, AST, ALT, check in rat liver tissue, to determine the rat hepatic oval cell activation model success. The second part. the extraction has been successfully set up eight rats hepatic oval cell activation in the model, separation after hepatic oval cells in vitro culture. Select four extraction of hepatic oval cells are cells induced factor EGF, another4points cells as normal controls, without using induction factors. Of culture cell detection of flow cytometry and fluorescence microscopy to identify the cells. The third part, the extraction process during induction differentiation respectively four generation times cells each generation in The Times of microRNAs (mir-21, mir-214), reverse transcription and real-time fluorescence quantitative detection of microRNAs.Results:1、2AAF+2/3ph1.2model through biochemical tests of ALT, AST, TbiL elevated, confirmed that can make the remaining seriously damaged liver cells, conform to the hepatic oval cell activation conditions.2. The expression of OV-6antigen of hepatic oval cells emit green fluorescent. The expression of CK19were antigen induced type bile duct epithelial cells glow red. With induction process of hepatic oval cells, the expression of CK19were rate is higher, prove hepatic oval cells to the higher level of bile duct epithelial cell differentiation, and hepatic oval cells OV-6sign expression gradually reduce, said the cells to differentiate into other cells.3. The epidermal growth factor (EGF) can induce hepatic oval cell proliferation differentiation for bile duct epithelial cells. Experiments prove that by using the epidermal growth factor (EGF) induced hepatic oval cells gradually become a kind of bile duct epithelial cells method is effective and can successfully induced into bile duct epithelial cells, and have the special surface marker phenotypic bile duct epithelial cells.4. By real-time fluorescence quantitative detection in hepatic oval cells differentiation into bile duct epithelial cells in the process, different time, different amount of mir-214expression in the differentiation level of cell level there was no significant difference. Each generation of cell of micrornas in the delta CT, q inspection (P>0.05), there was no statistically significant difference. Confirmed in the process of bile duct epithelial cells differentiation mir-214constant expression, no level difference.5. Through real-time fluorescence quantitative detection in hepatic oval cells differentiation into bile duct epithelial cells in the process, different time, different development levels of mir-21expression levels of cell differentiation to the P5cells and former P0, P1, P3generation cells was significantly difference, P5compared with P0, P1, P3cells (P<0.05), the difference was statistically significant. Confirmed in the process of bile duct epithelial cells differentiation mir-a constant expression, the early days of21no level difference adjustment, until the cell differentiation after nearly completely raised obviously.Conclusions:The experiment proved that the use of2-AAF+2/3PH method can effectively establish hepatic oval cell activationmodel, and by two step enzyme digestion successfully extracted from liver broken tissues of hepatic oval cells, the identification can be successfully induced hepatic oval cells induced into biliary epithelial cell by cell surface marker.Finally through real-time PCR, we found in the differentiation of hepatic oval cells in bile duct epithelial cells in the process, different generations, different levels of differentiation of the cells in the miR-21expression level of differentiation to P5cells and P0, P1,P3gener-ation cells showed significant difference. Confirmed in bile duct epithelial cells during the differentiation of miR-21early is a constant expre-ssion, no differences in the level ofregulation, until the cells increased significantly after nearly completely. Differentiation of hepatic oval cells in bile ductepithelial cells in the process, different generations, different levels of differentiation of the cells in the miR-214expression levels had no significant difference. Confirmed the expression of miR-214constant in bile duct epithelialcells during differentiation, no differences in the level of regulation. |
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