| Objective:To establish stable china minitype pig orthotopic liver transplantation acute rejection model by non-vein by -pass method.Method: Chinese miniature pigs includ Barman pigs buyed from experimental animal research center of Sichuan Academy of Medical Science and xizang pigs buyed from experimental animal research center of Southen Medical College,there are two groups,A group and B group,in A group:donors and receptors are all banna minitype pigs.in B group: donors are banna mintype pigs and receptors are Tibet minitype pigs,donors and receptors are matched randomly in each groups,during the experiment time,we have an intimate knowledge of pig liver dissection,all donors are accepted basis narcosis and all receptors accepted tracheal intubation +vein multiplicity narcosis in the experiment.Donor's liver is perfused by hepatic artery and portal vein.we determined liver biochemistry before operation and after operation one day,three day and six day.receptor's condition survival time and liver tissues of pathological change are observed.Result:32 orthotopic liver transplantation are carried out, average operational time is 139.61±14.27)min,average anhepatic phase is (20.28±3.43)min,the average volume of blood loss in the operation is 359.22±66.91ml,the average of blood transfusion is 410.06±51.13ml,no death occurred in the operation,one receptor died at the first day after operation because abdominal cavity haemorrhage,one died 3 days later because of lung infection,another two died 5days later and 7days later because of biliary fistula at the common bile duct stoma,no death occurred by portal vein and arterial stenosis.2,receptor 7 days survival rate after operation in A group is 90.9%,in B group is 70%,14days survival rate after operation in A group is 86.4%,in B group all receptors almost die during 7 days,14 days survival rate is 0,statistics significance is obverse in A group (P＜0.001).3.α=0.05 as the size of test,AST and Tbil comparison in the two groups are no statistics significance before operation(p＞0.05),but they all increase obviously after operation,especially in B group,AST total changes after operation in each group comparison are statistics significance(p＜0.001,F=15.11),Tbil total changes in each group comparison are statistics significance(p＜0.001,F=24.99),after operation AST,Tbil in two groups have different degree augmentation.In A group: AST increase slowly,the changes in 1day and 3day after operation comparison is no statistics significance(p=0.091),compared with 6day's has a obvious statistics significance(p=0.004),the 3 day's comparing with the 6day's is no statistics significance(p=0.82).Tbil increase slowly,the changes of Tbil in 1day compared with 3day's and 6day's after operation comparison is no statistics significance(p＜0.05),3day's compared with 6day's has no statistics significance (p＞0.05);in B group:the changes of AST after operation gradually increase,comparisons among different time have an obvious statistics significance (p＜0.001,F=73.35),the change in 1day compared with 3day's has no statistics significance(p＞0.05),compared with 6day's has statistics significance p＜0.001,3day's comparing with 6day's has statistics significance(p=0.01).the changes of Tbil after operation gradually increase,comparisons among different time have an obvious statistics significance(p=0.001,F=25.58),the change in 1day compared with 3day's has statistics significance p＜0.05,compared with 6day's has statistics significance(p＜0.05),3day's comparing with 6day's has statistics significance(p=0.025).4.pathological change:in A group shows ischemical reperfusion injury in Transplanted liver.receptors in B group occur obviously severely acute rejection after operation,many inflammatory cells aggregate hepatic tissue header domain.Some parts of hepatic lobule appear cellular necrosis.Conclusion:1.We have successfully established maturely stable china minitype pig orthotopic liver transplantation acute rejection model by non-vein by -pass method. 2.In A group receptor has an obviously longer survival time than that in B group, receptors in B group show severely acute rejection in transplanted liver,we successfully set up acute rejection model of orthotopic liver transplantation in minitype pigs between different closed population.3.Non-vein by -pass method is feasible to establish china minitype pig Orthotopic liver transplantation model.Objective To study receptor's autologous bone marrow stem cell which isolated culture and labeled by BrdU,which injected slowly into portal vein during the operation differentiation condition in Transplanted liver,and long-term survival influence for receptor,on the base of orthotopic liver transplantation in china miniature pigs.Method:establishing orthotopic liver transplantation acute rejection model in china miniature pigs by non -vein by-pass method,donor and receptors are banna pigs(buyed from experimental animal research center of Sichuan Academy of Medical Science)and Tibet pigs(buyed from experimental animal research center of Southen Medical College)respectively,we collected receptor's bone marrow in treatment group 14days ago before operation,and isolated,cultured bone marrow stem cells,at the same times we labeled these cells by BrdU,.experimentation set up the control group(we randomly get 8 examples from 10 examples in the first part B group as the control group)and the treatment group,we only practice orthotopic liver transplantation in the control group,the treatment group also accepts orthotopic liver transplantation,but we inject receptor's autologous bone marrow stem cell labeled by BrdU into portal vein during the opertation.14days 28days after operation,we anesthetize receptor and get a little of liver tissue,making patho-biopsy to observe transplanted cells's differentiation condition and function in Transplanted liver,at the same times we detect Liver function(AST,ALT)before operation and 1day,3days 6days after orthotopic liver transplantation,and observe receptor's general condition,survival time and pathological change of the liner tissue. Result:the cells labeled by BrdU masculine rate in treatment group at 14days and 28days after orthotopic liver transplantation shows 31.70±1.79%and 33.85±1.99% respectively,and there are no statistical significance between that two time point (P＞0.05).2.in the receptor's liver tissue the cells labeled by BrdU and albumin dyeing by immunohistochemistry SP method,express both masculine,the rate is 28.53±1.28%on 14days after operation,and 30.42±1.59%on 28days after operation,there are no statistical significance between them(P＞0.05).this shows that the receptor's bone marrow stem cells labled by BrdU can grow and differentiate into hepatic cell in liver microenvironment,and have biologic activity,sereting albumin;3.living condition:Median survival time in control group is 7days and 32days in treatment group,we can see that receptor in treatment group has a longer live time than that in the control group(P＜0.0001).in B group receptors die about lweek later,in A group receptor can live about lmonth;4.liver metergasis in two group:α=0.05 as the size of test.(1)After orthotopic liver transplantation,hepatic tissue incurs different degree damage,in treatment group this happens more lightly than that in control group, before operation we detected Tibet pig receptor's AST,ALT in the blood,and found the level of each them is almost the same,there are no statistical significance between them p＞0.05,but they have different degree augmentation after operation, AST total changes in two group comparison have statistics significance p=0.002,F=7.626,ALT total changes in two group comparison have obviously statistics significance p=0.002,F=8.877.(2)In treatment group:the changes among different time have statistics significance p＜0.001,F=123.15,the change of AST before operation compared with different time after operation has statistics significance p＜0.05.ALT in treatment group increase slowly,after operation 1day's compared with 3day's and 6day's has statistics significance p＜0.05,3day's compared with 6day's is no statistics significance p＞0.05.(3)In control group AST gradually increase after operation,comparison among different time have obviously statistics significance p＜0.001,F=46.64.1day compared with 6day's have statistics significance p=0.001,3day's compared with 6day's have statistics significance p=0.017.ALT gradually increase after operation,comparison among different time have obviously statistics significance p＜0.001,F=41.80,after operation 1day's compared with 3day's and 6day's has statistics significance p＜0.05.5,pathological change:after operation there has different degree immunological rejection in two groups,but receptor in control group express severely immunological rejection, Inflammatory reaction is more critical,a lot of lymphocyte aggregate in the header domain in the Transplanted liver,hepatic cell turbid and ballooning degeneration,we find large lobe cellular necrosis,in the treatment group pathological changes 3days after operation show:roughly normal liver tissue,hepatic tissue has a little congestion,a little inflammatory cell infiltrate around blood vessel,we can see lymphocyte infiltration below endothelial cell of small vein or hepatic vein,liver cell shows punctiform cellular necrosis,neutrophile granulocyte infiltrate.Conclusion:1.autologous bone marrow stem cell injected into portal vein during the orthotopic liver transplantation can prolong receptor's life;2.these stem cells can differentiate mature liver cell in the hepatic tissue and produce albumin;3.these differentiation cells can lessen immunological rejection and protect transplanted liver,and be the possible mechanism.
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