负载猪源性CTLA4Ig的猪胰岛细胞阻断直接识别途径在异种移植中的作用

目的：探讨负载供体源性CTLA4Ig的胰岛细胞通过阻断直接识别途径,诱导异种胰岛细胞抑制免疫耐受的可行性及有效性。方法：使用本实验小组前期构建的携带供体源性（猪）CTLA4-Ig的腺病毒载体（Adv-pCT-LA4-Ig）转染猪胰岛细胞,植入糖尿病大鼠肾包膜下,建立猪-大鼠异种胰岛移植模型,监测移植术后大鼠血糖变化及IL-4、γ-IFN的水平变化,检测移植物中pCTLA4-Ig、猪源性胰岛素（p-Insulin）表达情况。结果：1.大鼠血糖在移植术后即开始明显下降,于第3 d左右降至正常,三组大鼠血糖分别在移植术后6、7、35 d左右开始升高;2.三组大鼠术后胰岛平均存活时间为7.43±1.72 d、7.22±1.72 d、34.50±4.14 d,实验组胰岛细胞存活时间较对照组明显延长（P〈0.01）;3.细胞因子变化：（1）γ-IFN：对照组在移植术后第1 d开始轻度升高,于第3 d后开始明显升高,第7 d达到高峰,此后缓慢升高;实验组在移植术后第1～28 d波动较小,同术前相比无明显变化,在移植后第28 d开始上升,第35 d开始急剧上升;（2）IL-4：实验组在移植术后第1 d开始下降,第7 d达到谷值;对照组在移植术后第3～5 d轻微上升,在移植后第28 d开始下降至移植术前水平,第35 d开始急剧下降;4.病理学检查：实验组移植物无明显破坏,炎细胞浸润不明显;免疫组化可见大量pCTLA4-Ig、p-Insulin表达。结论：转染供体源性CTLA4-Ig的胰岛细胞通过阻断直接识别途径,可诱导异种胰岛细胞产生较长时间的免疫耐受。

Effect of Porcine Islet Loading Adv-porcine CTLA4-Ig Blocking the Direct Recognition Pathway in Xenotransplantation

Objective：To investigate the effectiveness,feasibility and possible mechanisms by transfected donor-derived CTLA4-Ig into islet cells,blocking the direct recognition pathway in inducing immune tolerance of islet xenotransplantation.Methods：The porcine islets transfected with porcine originated CTLA4-Ig gene（Adv-pCTLA4-Ig,by our experimental groups） were transplanted into kidney capsule of IDDM rats.Before the transplantation and post-operation,testing blood glucose and the IL-4 and γ-IFN levels were detected by the ELISA detection kit.The rats′ kidneys of the recipient rats were collected for pathology and immunohistochemistry in each group on the 18 days after transplantation.The expression of pCTLA4-Ig and p-Insulin were evaluated.Results：（1）The blood glucose levels decreased gradually to less than 11.1 mmol/L after about three days of the transplantation in each group and remained in normal range.The blood glucose levels increased on 6,7,35 days respectively.（2）The xenografts survival time in the three groups were 7.43±1.72 days,7.22±1.72 days and 34.50±4.14 days respectively.The grafts survival time were significantly prolonged in the experiment group compared with control group（P0.01）.（3）The changes of Cytokines in the transplanted rats： ①γ-IFN： in the control group,the level of increased gradually after the transplantation and peaked on the 7th day,and then increased slowly.In the experiment group,Minor fluctuations of the serum γ-IFN level could be observed in the 1-28 days after transplantation,and no significant change was detected compared with the preoperative level.The serum γ-IFN level began to rise on the 28th day after transplantation,and increased sharply on the 35th day after the transplantation.②IL-4： The serum IL-4 level of control group declined gradually after the transplantation.The serum IL-4 level of experiment group increased slightly in the 3-5 days after transplantation,and then fluctuated at this level;The IL-4 level began to decline on the 28th day after the transplantation.（4）The histochemical stain of the xenografts： Intact porcine islets and slightly inflammatory cells infiltration around the islets could be seen in the experiment group.P-Insulin and pCTLA4-Ig expression could be seen in the porcine islets in the experiment group.Conclusions：Donor originated（porcine） CTLA4-Ig gene-modified donor islet xenografts could induce tolerance in the rats by blocking the direct Recognition pathway.