Experimental Study On Biliary Defect And Repair Model Of South Yunnan Small Ear Pig

Objective(s):Preparation of human-derived acellular arterial matrix and detection of histocompatibility;establishment of a model of extrahepatic biliary tract defect in South Yunnan small ear pigs,and co-cultivation of porcine bile duct epithelial cells with human-derived acellular arterial matrix as tissue-engineered bile ducts to repair southern Yunnan Small ear pig liver with extrahepatic biliary tract defect.It will explore new ways and provide new applications and theoretical basis for the subsequent clinical practice of decellularized arterial matrix for repairing extrahepatic bile duct defects and benign stenosis.Methods:A two-step enzyme digestion method was used to prepare human-derived acellular arterial matrix,and its biocompatibility was examined using HE sections and Masson staining methods.The subject uses human decellularized arterial matrix and porcine bile duct epithelial cells to co-culture to prepare a new type of tissue engineered bile duct,and then establishes a model of extrahepatic bile duct defect in Yunnan South Small Ear Pig,and prepares the recellularized human decellularized artery Substrates were implanted into the Diannan small-ear pigs to repair their biliary defects.The research group randomly selected 28 closed groups of southern Yunnan small-eared pigs and randomly divided them into groups A,B,and C.Group A(n=12)used pure human-derived acellular arterial matrix patch to repair extrahepatic biliary tract Defects;Group B(n=12)using porcine bile duct epithelial cells and short segments(approximately 1.5 cm)of human-derived acellular arterial matrix composite culture patch to repair extrahepatic biliary tract defects;Group C(n=4)using porcine bile duct epithelial cells Repair the extrahepatic biliary tract defects with a long-term(about 3.0cm)human acellular arterial matrix composite culture patch;observe the general conditions of experimental animals closely after operation,and at the planned time points(1 week,2 weeks,4 weeks and 8)Week)Each group of 3 animals in group A,3 groups of animals in B and 1 group of animals underwent magnetic resonance biliary imaging examination,liver function and blood routine examination,and were sacrificed for dissection to observe whether there was bile leakage in the abdominal cavity of the experimental animals,Abdominal infection and intestinal adhesions.After further dissection,free the common bile duct to observe whether the common bile duct has stenosis and obstruction,and whether the liver has cholestasis;cut the common bile duct and cut it longitudinally to observe whether there is stenosis,scar hyperplasia,stone formation,bile leakage,tumor occurrence,etc.Take the repaired common bile duct tissue for further laboratory testing.For the experiment of acellular arterial matrix histocompatibility,the research team randomly selected 8 small-eared Yunnan pigs from the Animal Experimental Center of Kunming Medical University,with male and female limits,weighing about 20 kg.No food or drink 12 hours before surgery The experimental animals were implanted with a human-derived acellular matrix patch under the skin of the inner thigh area of the right lower limb.The healing of the surgical port was observed closely after the operation.Two animals were planned to be killed in the first,second,fourth,and eighth weeks.Whether there were redness,empyema,fluid accumulation,and poor healing in the mouth,and the embedded tissue was removed for routine sectioning,HE staining,and Masson staining to observe the morphological characteristics of the tissue.Results:In the study,we successfully prepared human-derived acellular arterial matrix and tested histocompatibility,and found that it showed extremely low immunogenicity in both biliary and non-biliary tissues.Successfully established a stable model of extrahepatic biliary tract defect in South Yunnan small-ear pigs,and used human-derived acellular arterial matrix combined with porcine bile duct epithelial cells to repair its extrahepatic biliary duct defect.Among them,group A used a pure human-derived acellular arterial matrix patch to repair the extrahepatic bile duct defects of experimental animals.From week 2 on,experimental animals have mild to severe jaundice,and magnetic resonance cholangiography shows biliary tract There was mild to severe obstruction,with cholangitis and bile duct dilation inside and outside the liver.The reason for the analysis may be that the acellular matrix was immersed in bile and began to degrade in the second week.After the degradation,the bile corroded the surrounding tissues and caused scar hyperplasia,causing obstruction,which caused a series of consequences including obstructive jaundice and infection.Three animals wishing to observe for 8 weeks showed mental deterioration and poor diet since the 7th week,and died on the 52nd,54th and 55th days after surgery.The experimental animals in group B used pig bile duct epithelial cells and a short segment(about 1.5 cm)of human-derived acellular arterial matrix composite growth patch to repair extrahepatic biliary tract defects at weeks 1,2,4,and 8 weeks.All the experimental animals survived to the planned time point naturally,and no obvious biliary obstruction was found in liver function test.Magnetic resonance cholangiography showed no obvious bile leakage and biliary strictures.Our decellularized vascular stent successfully drained bile to the duodenum for 8 weeks.After implanting GFP-carrying bile duct cells into the acellular arterial matrix,histology and immunofluorescence detection can still detect GFP-positive bile duct cells up to the 8th week,which shows that our bile duct cells have been successfully implanted.This suggests that the recellularization of the human decellularized arterial matrix has a certain protective effect,and it also confirms from the side that the human decellularized arterial matrix with composite biliary epithelial cells has a more optimistic prospect in the application of biliary tract repair.Considering certain liver transplantation or severe iatrogenic biliary tract injury,we extended the length of the patch to conduct the experiment in group C,but the results were not as good as in group B.One animal in group C is planned to observe 8 weeks The experimental animals began to suffer from mental and appetite deterioration at week 7 and mild yellowing of skin and sclera.The animal was sacrificed 8 weeks after the scheduled time.The animal was dissected and found to have scar hyperplasia of the common bile duct,blocked bile duct,and mild bile duct.Expansion occurs with some intrahepatic biliary dilatation.The reason for the analysis may be that the patch is too long,and the repair time of the host's biliary tract is prolonged,and the long human decellularized arterial matrix composite bile duct epithelial cells is not enough to provide sufficient transition time.Conclusion(s):The human-derived arterial decellularized matrix prepared by us has exhibited excellent characteristics in the biliary and non-biliary tract systems of Diannan small-ear pigs.No acute or subcutaneous and biliary tract patch repair models of Diannan small-ear pigs' biliary tract defects were found.Chronic rejection reaction suggests that the human-derived acellular arterial matrix is extremely immunogenic and has good tissue compatibility with experimental animal organisms.We have successfully established a model of extrahepatic biliary tract defect and repair in South Yunnan small-eared pigs.Compared with the simple decellularized arterial matrix and the decellularized arterial matrix of the long composite bile duct epithelial cells,the short decellularized arterial matrix composited with porcine bile duct epithelial cells is better in repairing extrahepatic biliary tract defects.