【作者】 潘明新；

【导师】 高毅；

【作者基本信息】 第一军医大学 ， 肝胆外科， 2007， 博士

【摘要】 目的:通过非静脉转流方式建立稳定成熟的小型猪原位肝移植急性排斥反应模型。方法:小型猪包括两种小型猪:版纳小型猪(由四川医学科学院实验动物研究所提供);西藏小型猪(由南方医科大学动物研究所提供)。实验分为A、B两组,A组:供体、受体均为中国版纳小型猪,B组:版纳猪作为供体,西藏小型猪作为受体,每一组中的供体和受体随机匹配,在充分熟悉猪肝脏的解剖特点的基础上,进行原位肝移植。在整个实验中供体采用基础麻醉,通过肝动脉及门静脉双灌注方法进行供肝灌注,受体采用气管插管+静脉复合全麻醉。手术前术后测定肝功能(AST,Tbil)、术后观察肝移植后猪的一般状况,存活时间和肝脏组织的病理变化。结果:1、共进行小型猪原位肝移植手术32例,平均手术时间(139.61±14.27)min,平均无肝期(20.28±3.43)min,术中平均失血量359.22±66.91ml,平均输血量410.06±51.13ml,无术中死亡,1例因腹腔出血术后1天死亡,因胆总管吻合口胆瘘术后第5天死亡1例,1例术后3天因肺内感染死亡,另1例术后第7天也因肺内感染死亡。受体死亡后进行解剖未见门静脉及肝动脉狭窄的情况。2、各组受体术后7d成活率分别为A组90.9%,B组70%,14天存活率A组为86.4%,B组受体均在1周左右死亡,14天存活率为0,A组生存时间明显比B组延长(P＜0.001);3、α=0.05为检验水准,在术前两组血清AST、Tbil值的比较尚无统计学差异p＞0.05,A组和B组中术后的AST总体变化比较有显著的统计学差异(p＜0.001,F=15.11);A组和B组中术后的Tbil总体变化比较有显著的统计学差异(p＜0.001,F=24.99);术后AST、Tbil在两组中都有升高,A组中AST升高的缓慢术后1天和术后3天比较没有统计学意义(p=0.091),与术后6天比较有统计学意义(p=0.004),术后3天和术后6天的比较没有统计学意义(p=0.82),Tbil升高的缓慢术后1天分别与术后3天、6天比较有统计学意义(p＜0.05),术后3天和术后6天比较没有统计学差异(p＞0.05);B组中术后AST逐渐升高,不同时间点的变化有显著的统计学意义(P＜0.001,F=73.35),术后1天和术后3天的比较无统计学意义(p＞0.05),与术后6天的比较有统计学差异(p＜0.001),术后3天的和术后6天的比较有统计学差异(p=0.01)。Tbil也是逐渐性升高不同时间点的比较有显著的统计学意义(P=0.001,F=25.58),术后1天的分别与术后3天、6天的比较有统计学差异(p＜0.05),术后3天与术后6天比较有统计学意义(p=0.025)。4、病理变化:术后A组移植肝脏中出现了缺血再灌注损伤的病理表现,术后B组移植肝脏中出现严重急性排斥反应,肝组织汇管区大量炎症细胞聚集,部分肝小叶出现坏死。结论:1、成功地建立了成熟稳定的非静脉转流方式的中国小型猪原位肝移植急性排斥反应的模型;2、A组的受体在肝移植后的生存时间明显长于B组中的受体,B组受体移植的肝脏中出现了严重的急性排斥反应,本研究成功的建立了不同封闭群小型猪之间的异体原位肝移植的急性排斥反应模型;3、非静脉转流方式在小型猪原位肝移植中是可行的。目的:在已建立的小型猪原位肝移植急性排斥反应模型的基础上,研究在术中经门静脉输注经BrdU标记的自体骨髓干细胞在移植肝中的分化状况和对受体长期存活影响。方法:非静脉转流的方式建立小型猪的原位肝移植急性排斥反应的模型,供体猪为版纳小型猪(由四川医学科学院实验动物研究所提供),受体猪均为西藏小型猪(由南方医科大学动物研究所提供),实验分为两组,对照组(在第一部分实验中,从B组的10例受体中随机抽取8例作为第二部分的对照组)和处理组,提前抽取处理组受体猪的骨髓血,分离培养获得骨髓干细胞并经BrdU标记,对照组仅行原位肝移植,处理组在原位肝移植的基础上,在术中经门静脉输注标记好的受体自身骨髓肝细胞,术后14天、28天,取病理组织活检观测移植细胞的分化状况和功能,同时在受体麻醉后开腹术前,术后1天、3天、6天采静脉血检测肝功能的变化(AST、ALT),并观测猪的一般状况和生存时间及病理变化。结果:1、处理组中BrdU标记细胞的阳性率在移植后14天、28天分别为31.70±1.79%,33.85±1.99%,两者之间比较没有统计学差异(P＞0.05);2、BrdU标记细胞和白蛋白染色双阳性率在术后14天、28天分别为28.53±1.28%,30.42±1.59%,两者之间无统计学意义(P＞0.05)。这说明BrdU标记的受体自身骨髓干细胞在移植的肝脏微环境中发育分化为肝细胞,并具有生物学活性,能够分泌白蛋白;3、生存状况:两组中位生存时间(Mediansurvival time,MST)分别为:对照组7d,处理组32d,处理组明显比对照组的生存时间延长P＜0.001。对照组中的受体一般都在1周左右死亡,而处理组中的受体大多数能够存活1个月左右;4、肝功能变化:α=0.05为检验水准,(1)术前两组血清AST、ALT值的比较尚无统计学差异(p＞0.05),处理组和对照组中AST总体变化比较有显著的统计学差异(p=0.002,F=7.626);处理组和对照组中ALT总体变化比较有显著的统计学差异(p=0.002,F=8.877);(2)术后AST、ALT在两组中都有升高,处理组中AST在不同时间点的变化有统计学意义(p＜0.001,F=123.15),术前AST变化分别与术后不同时间点的变化比较有统计学意义(p＜0.05);处理组中ALT升高的缓慢术后1天分别与术后3天、6天比较有统计学意义(p＜0.05),术后3天和术后6天比较没有统计学差异(p＞0.05);(3)对照组中AST术后逐渐升高,不同时间点比较有显著统计学意义(P＜0.001,F=46.64).术后1天与术后6天的比较有统计学差异(p=0.001),术后3天和术后6天比较有统计学差异(p=0.017).ALT也是逐渐性升高不同时间点的比较有显著的统计学意义(P＜0.001,F=41.80),术后1天的分别与术后3天、6天的比较有统计学差异(p＜0.05);5、组织病理变化:术后两组均出现不同程度的免疫排斥反应,对照组在术后3天移植的肝脏中出现汇管区大量炎症细胞聚集,主要是淋巴细胞,炎症反应重,肝细胞浑浊,气球样变性并出现不同程度的点状、灶状坏死,而处理组在术后3天病理活检见:大致呈正常肝脏病理,肝组织轻度淤血,血管周围少量炎性细胞浸润,少数汇管区小静脉或肝静脉内皮细胞下淋巴细胞浸润,肝细胞点状坏死,中性粒细胞浸润。结论:1、自体骨髓干细胞延长了受体的存活时间;2、能够在移植肝脏中分化为肝细胞,并产生白蛋白;3、减轻免疫排斥反应,保护移植的肝脏免受损害,门静脉输注自体骨髓干细胞可能是减轻排斥反应、延长受体生存时间的机制。更多还原

【Abstract】 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.更多还原