课题背景：免疫抑制剂的应用，虽然有效地抑制了急性排斥反应，但长期应用免疫抑制剂必然会给移植患者带来许多副作用和潜在的危险性。因此，诱导受者对供者器官特异性免疫耐受是解决排斥反应最理想的措施。国内外学者对诱导免疫耐受的尝试甚多，但结果均不甚理想，机制尚不明确，更没有成熟可应用于临床的方案。因此，如何成功地在成年大动物和临床诱导免疫耐受成为免疫学家和移植学家的主攻方向和目标。

应用要点：实验采用基因纯合度高的中国版纳小型猪，采用非转流条件下的原位肝移植手术方法，建立小型猪原位肝移植模型。先前已成功建立小动物移植模型并验证了供体凋亡细胞预处理受者能够诱导免疫耐受，并提出了一个新的观点：肝脏是特异性吞噬凋亡细胞的场所，凋亡细胞有局部免疫抑制的作用，吞噬了凋亡细胞的肝脏抗原递呈细胞在局部免疫抑制的环境下递呈抗原给T细胞，而诱导对被吞噬抗原的免疫耐受。现进行大动物实验来验证作者的发现，同时进一步探索凋亡细胞诱导免疫耐受的机制。

同行评价：目前诱导供者特异性免疫耐受已经成为免疫学家的重大研究课题。文章作者能够抓住这一国际前沿课题开展实验研究，进行了大量的动物实验，成功地诱导了大鼠的供者特异性免疫耐受，并在此基础上进行创新——开展大动物猪的实验，提供了新的思路，对今后的实验具有指导作用。

摘要

背景：作者先前已成功建立小动物移植模型，并验证了受者经供体凋亡细胞预处理后能够诱导免疫耐受。
目的：验证60Co γ射线体外处理后的供者淋巴细胞预输注诱导猪肝移植特异性免疫耐受的可能性，为不作常规免疫抑制处理成年大动物的移植免疫耐受诱导提供可行的途径。
设计、时间及地点：于2004-06/2007-02在南方医科大学南方医院动物研究所完成随机对照动物实验。
材料：版纳小型猪16只为供体组，西藏小型猪16只为受体组，五六月龄，体质量15~17 kg，雌雄不拘，共行原位肝移植手术16只次。
方法：将受体猪按随机数字表法分为2组（n=8），空白对照组受体猪无特殊处理；供体淋巴细胞预处理组受体猪在术前7 d经耳静脉注射60Co γ射线处理过的供体淋巴细胞。
主要观察指标：应用AnnexinV/PI双标法检测60Co γ射线处理后的淋巴细胞凋亡率，并与正常淋巴细胞进行对照。观察两组受体猪移植术后的存活时间，术后检测肝功能指标及肝脏病理变化。
结果：成功建立非转流小型猪原位肝移植模型16例。①60Co γ射线体外处理后的淋巴细胞凋亡率明显高于正常淋巴细胞（P < 0.01）。②两组受体猪移植术后中位存活时间相同。③移植术后3 d，两组病理活检均呈急性中、重度排斥反应；术后6 d，两组均呈急性重度排斥反应。④两组移植术后肝功能检测血清丙氨酸转氨酶、天冬氨酸转氨酶活性及总胆红素、总蛋白及白蛋白含量测定结果均为肝细胞破坏表现，两组无明显差异（P > 0.05）。
结论：60Co γ射线体外照射可以诱导淋巴细胞凋亡，60Co γ射线体外处理过的淋巴细胞预输注未能够诱导小型猪同种异体肝移植特异性免疫耐受。
关键词：肝移植；免疫耐受；脱噬作用；猪，雏型；疾病模型，动物；器官移植

谢金敏，高毅，潘明新，孔凡东，姚坤厚，王琼.供体凋亡淋巴细胞预输注诱导猪肝移植的免疫耐受[J].中国组织工程研究与临床康复，2008，12(18):3418-3422 [www.zglckf.com/zglckf/ejournal/upfiles/08-18/18k-3418(ps).pdf]

Immune tolerance to porcine liver transplantation induced by pre-infusion of 60Coγ-treated donor lymphocyte

Abstract

BACKGROUND: In previous studies we have successfully established small animal models of transplantation, and validated immune tolerance of recipient can be induced by preconditioning of donor apoptotic cells.
OBJECTIVE: To investigate the influence of preoperative transfusion of lymphocyte from in vitro 60Coγ-treated donor porcine on allograft survival of orthotopic liver transplantation, and to develop new protocols for transplantation tolerance induction in adult animals under no routine immunosuppression.
DESIGN, TIME AND SETTING: The randomized controlled animal trial was performed at Research Institute of Animal, Nanfang Hospital of Southern Medical University from June 2004 to February 2007.
MATERIALS: Sixteen Banna Mini-pigs were used as donors and sixteen Tibet Mini-pigs as recipients, 5-6 moths old and 15-17 kg, irrespective of gender. Orthotopic liver transplantation was performed for 16 animals.
METHODS: Donor pigs were randomly divided into two groups (n=8). Blank control group was not given any specific treatment. In lymphocyte preconditioning group, animals were injected with 60Co γ-treated donor lymphocyte preoperatively 7 days.
MAIN OUTCOME MEASURES: Apoptosis rate of lymphocyte treated by 60Coγwas measured by AnnexinV/PI double staining method, and compared with normal lymphocyte. Survival time of pigs in two groups after tranplantation was observed, and liver function parameters and liver pathological alterations were detected.
RESULTS: Sixteen pig models of orthotopic liver transplantation were successfully established. 60Coγ-treated lymphocyte apoptosis rate was significantly higher than that of normal lymphocyte (P < 0.01). Liver grafts of two groups had a identical median survival time. Liver graft biopsy of two groups showed pathological mild or serious acute rejection 3 days after operation, and serious acute rejection on the 6th day. The liver function parameters such as serum alanine transaminase, aspartate aminotransferase activity, total bilirubin, total albumin, and albumin showed liver pathological changes in two groups after the transplantation. But there were no significant differences between two groups (P > 0.05).
CONCLUSION: 60Co γirradiation can induce lymphocyte apoptosis in vitro. Immune tolerance cannot be induced by preoperative transfusion of donor 60Co γ-treated lymphocyte in mini-pigs undergoing liver allograft.

Xie JM, Gao Y, Pan MX, Kong FD, Yao KH, Wang Q. Immune tolerance to porcine liver transplantation induced by pre-infusion of 60Coγ-treated donor lymphocyte.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(18):3418-3422(China)
[www.zglckf.com/zglckf/ejournal/upfiles/08-18/18k-3418(ps).pdf]