经hCTLA4-Ig基因转染的猪胰岛细胞在小鼠体内的存活与功能状况研究  

Effect of AVV-hCTLA4-1g transfection of porcine islets on survival of grafts in mice

作　　者：莫朝晖[1] 王维[1] 刘涛[1] 曾秋华[1] 吴小兵[2] 邢晓为[1] 金萍[2] 卢光琇 

机构地区：[1]中南大学湘雅三医院细胞移植与基因治疗中心内分泌科,长沙410013 [2]中国预防医学科学院病毒基因载体国家重点实验室 [3]中南大学湘雅三医院人类干细胞研究国家工程中心,长沙410013

出　　处：《中华器官移植杂志》2007年第6期323-326,共4页Chinese Journal of Organ Transplantation

基　　金：国家自然科学基金(39994013);卫生部临床重点项目(20012841)

摘　　要：目的研究将人细胞毒T淋巴细胞相关抗原4免疫球蛋白(hCTLA4-Ig)基因转染至猪胰岛细胞后,再将其移植至小鼠体内的存活及功能状况。方法采用腺相关病毒载体(AAV)介导hCTLA4-Ig基因体外转染新生猪胰岛细胞(NIPs),再将转基因的NIPs移植至构建了人免疫系统的SCID糖尿病小鼠左肾被膜下。逆转录聚合酶链(RT-PCR)反应和免疫荧光染色法检测转染后hCTLA4-Ig基因的表达状况;观察小鼠移植转基因NIPs后的生存时间;移植物免疫组织化学分析及酶联免疫(ELISA)法测定受者血清中细胞因子的水平。结果NIPs经转染后,可检测到hCTLA4-Ig基因及其蛋白表达,且葡萄糖刺激胰岛素释放试验与未转染的对照组比较,差异无统计学意义(P>0.05);糖尿病小鼠经转基因的NIPs移植后,生存时间及血糖维持正常的平均时间分别为(72.5±30.6)d和(59.1±24.0)d,较未转染的对照组(26.9±6.9)d和(12.7±3.3)d显著延长(P<0.01);免疫组织化学染色检测移植后不同时间(15、30、90d)的移植物组织,可见转基因细胞移植部位有完整的胰岛细胞,而对照组胰岛细胞破坏、消失,周围可见较多的炎性细胞浸润;且转基因细胞移植的小鼠血清白细胞介素2(IL-2)、γ干扰素(IFN-γ)、肿瘤坏死因子α(TNF-α)的水平明显低于对照组(P<0.05)。结论AAV介导hCTLA4-Ig基因体外转染猪胰岛细胞后再移植至受者体内,可提高受者的免疫耐受水平,延长胰岛细胞在异种受者体内的存活时间,而胰岛细胞的内分泌功能不受明显影响。Objective To investigation the role of AAV-hCTLA4-Ig transfection of newborn porcine islets （NPIs） on the survival of the xenogeneic host. Methods Cultured NPIs were transfected with AAV- hCTLA4-Ig. The expression of CTLA4-Ig was assayed by RT-PCR and immunocytochemistry in these cells （in the experimental group and the control group）, then these cells were transplanted into the left renal sub-capsule of human lymphocyte-reconstituted SCID mice with drug-induced diabetes. The histological examination of grafts was performed to evaluate the grafts morphology and the level of serum cytokine assayed by ELISA and the survival time of recipients was recorded. Results CTLA4-IgmRNA and protein were detected in the experimental group by RT-PCR and immunocytochemistry. There was good response of glucose-stimulated insulin release in the experimental group, with no significant difference compared with the control group （P〉0. 05）. After transplantation, in the experiment group the survival and normoglycemia duration were （72. 5 ± 30. 6） d and （59. 1 ± 24. 0） d respectively, significantly longer than in the control group （26. 9 ± 6. 9） d and （12. 7 ± 3. 3） d respectively and vacant control group （18. 6 ±5. 8） d and 0 d respectively. Histological examination, on the 15th, 30th and 90th day post transplantation, revealed that intact NPIs could be detected in the transplantation position in the experiment group, while NPIs were destroyed, even disappeared with markedly lymphocytes infiltration in the control group. The levels of IL-2, IFN- γ and TNF-α in the experimental group were significantly lower than those in the control group （P〈0. 05）. Conclusion AAV mediated hCTLA4-Ig expression in NPIs could induce immunologic tolerance in xenotransplantation and prolong survival time of islet grafts in the host, while the endocrine function of NPls were not affected significantly.

关 键 词：基因转移技术 猪 胰岛 移植 异种 免疫耐受 

分 类 号：R657.5[医药卫生—外科学] R587.1[医药卫生—临床医学]