CTLA4Ig基因修饰BMSCs对异种胰岛移植排斥反应的影响

Effects of CTLA4Ig modified bone marrow mesenchymal stem cells on the rejection of rat islet xenografts

目的探讨细胞毒性T淋巴细胞相关抗原4免疫球蛋白(CTLA4Ig)基因修饰骨髓间充质干细胞(BMSCs)在异种胰岛移植排斥反应中的作用。方法构建携带CTLA4Ig基因的重组腺病毒载体,用其感染BMSCs并和人胰岛细胞移植到糖尿病大鼠肾包膜下,建立人-大鼠异种胰岛移植模型。观察胰岛移植后糖尿病大鼠血糖变化、生存情况以及移植物病理形态学改变,检测移植物胰岛素和CTLA4Ig的表达以及移植糖尿病大鼠白细胞介素2(IL-2)和肿瘤坏死因子(TNF-α)的水平变化。结果①糖尿病大鼠血糖在移植后2～3 d恢复正常,对照组血糖平均在移植后7 d升高,单纯干细胞组和干细胞转染组血糖分别在22 d和46 d升高。②对照组、单纯干细胞组和干细胞转染组移植物存活时间分别为(10±2.8)d、(27±6.5)d和(52±8.7)d,各组间比较差异有统计学意义(F=6.459,P<0.05);移植大鼠生存时间分别为(25±7.6)d、(55±9.8)d、(98±16.3)d,各组间比较和移植物存活时间相似(F=7.365,P<0.05)。③对照组在移植后1周内,IL-2、TNF-α的水平均急剧上升,较移植前显著升高(P<0.01)。④各治疗组移植物见成片的胰岛细胞团,未见淋巴细胞浸润,转染组移植物可见胰岛素和CTLA4Ig的表达。结论 CTLA4Ig基因修饰骨BMSCs可抑制异种胰岛移植排斥反应,延长胰岛移植物的存活时间。

Objective To study the effects of cytotoxic Tlymphocyte associated antigen 4 immunoglobulin（CTLA4Ig） modified bone marrow mesenchymal stem cells（BMSCs） on the rejection of rat islet xenografts.Methods The recombinant adenoviruses containing CTLA4Ig gene was constructed and BMSCs were infected with the recombinant adenoviruses.Human islets and infected BMSCs were transplanted under the left kidney capsule of diabetic rats.The blood sugar of transplantation rats was measured and the survival of transplantation rats and grafts were observed after transplantation.The morphological changes of grafts were observed.Expression of insulin and CTLA4Ig were detected by immunohistochemical staining and cytokines were quantified by ELISA.Results ① The blood glucose of transplantation rats decreased to normal level on day 2-3 post-transplantation.The average level blood glucose of control group（A）,BMSCs group（B）,transfected BMSCs group（C） increased on day 7,22,46,post-transplantation respectively.② The grafts of group A,B and C survived for 10±2.8,27±6.5 and 52±8.7 days respectively.The survival of grafts in group C was significant longer than that in group A and B respectively;The survival of group B were significantly prolonged compared with group A（F=6.459,P〈0.05）.The survival of transplantation rats were 25±7.6,55±9.8 and 98±16.3 days in group A,B and C respectively.The survival of transplantation rats compared each other among three groups were same as the survival of grafts（F=7.365,P〈0.05）.③In control animals（group A）,Serum TNF-α and IL-2 concentration were elevated to a high level within seven days post transplantation and significantly increased compared with that before transplantation（P〈0.01）.④Hematoxylin-eosin staining of grafts showed a lot of islets under the kidney capsule of transplantation rats,no inflammatory cell infiltrate and immunohistochemical staining of grafts demonstrated expression of insulin protein at islets in group B and C.These grafts positively stained for CTLA4Ig in group C.Conclusion CTLA4Ig gene modified bone marrow mesenchymal stem cells c could inhibit the rejection of rat islet xenografts and prolong the survival time of islet xenografts.