| Objective To construct an animal model which hepatitis B virus X protein(HBx)directly effect on hepatic progenitor cells(HPCs),and investigate its potential mechanisms involved in epithelial-mesenchymal transition(EMT).Methods Screening of stably transfected cell lines by lentivirus.The Kunming mice were given 2 m L/L carbon tetrachloride in oil solution by gavage twice a week,then the transfected HPCs which were transfected by recombinant lentiviral vectors stable expression HBx gene as well as empty vectors separately infused to the mouse from the portal vein and and have a partial hepatectomy at the same time.In postoperative 3 days,5 days,7days,14 days,21 days and 28 days,the mice were sacrificed and the livers were removed.Fluorescence quantitative PCR was used to detect the m RNA expression level of HBx,Immunohistochemistry was used to detect the exogenous cells survival in vivo.Fluorescence quantitative PCR was used to detect the m RNA expression level of E-cadherin,N-cadherin,vimentin and CK18,and Western blotting was used to detect the protein expression level of E-cadherin,N-cadherin,vimentin and CK18.Results 1.14-19 cells were detected by inverted microscopy,NC-14-19 cells and HBx-14-19 cells were detected by fluorescence microscopy and GFP was used to check transfection efficiency.After 3 passages,the expression level was still more than 90%,which confirmed that the cells could express the GFP gene stably;2.The expression of HBx gene by fluorescence quantitative PCR assay showed that the expression of HBx gene in HBx-14-19 cells was significantly higher than that of 14-19 cells and NC-14-19 cells(P <0.05).HBx-14-19 cells and NC-14-19 cells were successfully constructed.3.Frozen sections of different days of liver tissues in postoperative were detected by fluorescence microscopy.The experimental group and the control group had the expression of green fluorescence in different days after operation,and the expression increased with the days after operation.The expression level of HBx m RNA with different days in postoperative by real-time fluorescence quantitative PCR found that the experimental group compared with control group,the expression of 3d,5d,7d,14 d,21d,28 d of liver tissue HBx m RNA were higher(P<0.05),with the progress of time after operation,the contents of HBx increased significantly.Animal model was successfully constructed.5.Compared with the negative control group,the experimental group in different days of hepatic progenitor cells injected with HBx gene,E-cadherin m RNA levels were decreased(P<0.05),N-cadherin and m RNA in different days showed an increasing trend,after 3~5 days increased significantly(P<0.05).The expression of Vimentin m RNA was increased,and the expression of 7d was highest(P<0.01).CK18 m RNA after operation in the experimental group compared with the control group,the overall downward trend,but after third days the expression increased significantly(P <0.05),after 14 d and 28 d decreased most significantly(P<0.01),the rest were decreased(P <0.05).6.The expression level of EMT related markers E-cadherin,N-cadherin,Vimentin and CK-18 protein with different days in postoperative by Western blotting.Compared with the negative control group,the experimental group in different days of hepatic progenitor cells injected with HBx gene,E-cadherin protein content showed a declining trend,14 d decreased significantly(3d,5d,7d,14 d for P<0.01,21 d,28d for P <0.05),and N-cadherin protein in operation after the overall upward trend(P<0.05).The expression of Vimentin protein was increased,and the expression of protein was significantly increased at 7d(7d,14 d,21d for P <0.01,the others P <0.05).CK18 protein showed a downward trend(3d,5d for P<0.05,the others P <0.01).Conclusion NC-14-19 cells and HBx-14-19 cells could express the GFP gene stably.Animal model of stable expression of HBx gene was successfully constructed.HBx plays an important role in the regulation of EMT of HPCs in vivo. |
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