Experimental Study On Repairing Biliary Tract Defect Of Diannan Porcine By Heterogeneous Acellular Artery Matrix

Objective(s):The biliary defect was repaired by decellularized arterial matrix covered with lentivirus-labeled bile duct epithelial cells.Specimens were obtained at different time points,and the growth of the cells in the biliary tract after repair was observed.It provides a new theoretical and applied basis for the clinical application of acellular arterial matrix to repair biliary defects.Methods:Porcine biliary epithelial cells were labeled with pHBLV-CMV-mcs-EFl-ZsGreen-T2A-luc lentivirus and cell growth was measured by CCK-8 method and growth curves were plotted.The labeled porcine biliary epithelial cells were cultured in combination with human acellular matrix to prepare composite epithelial tissue and implanted into the animal to repair biliary defects.Six Minnan small ear pigs were randomly divided into experimental group and control group,with 3 rats in each group.The experimental group used lentivirus-labeled bile duct epithelial cells and human acellular decellularized matrix matrix growth patch for extrahepatic biliary defect repair.The group was repaired with a human decellularized artery matrix patch.One experimental group and the control group were sacrificed at 1 week,2 weeks,and 4 weeks after operation.The repaired common bile duct tissues were observed by routine sectioning and HE staining,and the quantitative analysis was performed by fluorescence quantitative PCR and Western Blot.Changes in the expression of related indicators.The growth of the cells in the biliary tract after repair was observed in the acellular matrix.Results:Compared with the unlabeled control group,pHBLV-CMV-mcs-EF1-ZsGreen-T2A-luc-labeled bile duct epithelial cells showed no significant difference in cell proliferation and growth,and cell growth was continuous,and its growth curve showed a gradual increase.It did not show growth restriction after a period of growth and resulted in an "S" shape of the growth curve,which was consistent with the direct observation during cell culture.One week after implantation in the acellular arterial matrix,the biliary epithelial cells were observed by scanning electron microscopy.A small number of biliary epithelial cells were attached to the surface of the substrate and the collagen fibers exposed on the inner surface of the matrix.After 2 weeks of implantation,scanning electron microscopy showed that the bile duct epithelial cells partially covered the endometrial surface of the stroma,but most of the decellularized arterial stroma remained uncovered.After 3 weeks of implantation,scanning electron microscopy revealed that the bile duct epithelial cells covered most of the endometrial surface of the stroma.After 4 weeks of implantation of the bile duct epithelial cells,the inner membrane surface of the matrix of 1x1 cm2 was covered substantially,and the collagen fibers exposed on the inner membrane surface of the matrix were not observed.The composite tissue planted for 4 weeks was selected as the experimental group to repair the patch.The expression of GFP gene in the experimental group increased with time,and no GFP gene was detected in the control group.The expressions of CK7,CK19 and GGT genes in the experimental group and the control group increased with time.At the same time point,the relative expressions of CK7,CK19 and GGT genes in the experimental group and the control group were P<0.05,the difference was statistically significant.The relative expression of the experimental group was higher than that of the control group.The expression of GFP protein in the experimental group increased with time,and no GFP protein was detected in the control group.The expressions of CK7,CK19 and GGT protein in the experimental group and the control group increased with time.At the same time point,the relative expression of CK7,CK19 and GGT protein in the experimental group and the control group were P<0.05,the difference was statistically significant.The relative expression of experimental histones was higher than that of the control group.Conclusion(s):There is no significant difference between the lentivirus-labeled bile duct epithelial cells and the blank cells,and the markers can be long-term effective,and can stably express GFP for a long time,and can effectively trace.the target cells in living animals.The human heterogeneous acellular arterial matrix has good histocompatibility with the extrahepatic biliary tissue of Minnan small ear pig.After repairing the extrahepatic biliary defect of Minnan small ear pig,it can be used as extracellular scaffold to partially reconstruct the defect biliary tissue.The biliary epithelium regenerates and covers the surface of the decellularized motility matrix.Bile duct epithelial cells can accelerate the repair process of defects,and the decellularized arterial matrix covering bile duct cells plays an active role in the whole repair process.