AAV体外介导hCTLA4-Ig在新生猪胰岛细胞中的表达与生物学活性  

Expression of hCTLA4-Ig mediated by adeno-associated virus in porcine islets and their biological activity

作　　者：莫朝晖[1] 王维[1] 刘涛[1] 曾秋华[1] 吴小兵 谢艳红[3] 

机构地区：[1]中南大学湘雅三医院细胞移植与基因治疗研究所,长沙410013 [2]中南大学中国预防医学科学研究所病毒基因工程国家重点实验室,北京100052 [3]中南大学湘雅三医院内分泌科,长沙410013

出　　处：《中南大学学报（医学版）》2007年第1期36-40,共5页Journal of Central South University ：Medical Science

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

摘　　要：目的:探讨AAV-hCTLA4-Ig体外感染猪胰岛细胞后,hCTLA4-Ig基因的表达能力及生物学活性。方法:腺相关病毒载体(AAV)介导hCTLA4-Ig体外转染新生猪胰岛细胞(NIPs),用RT-PCR和免疫细胞化学法检测胰岛细胞内hCTLA4-Ig mRNA及蛋白质水平的表达。转染后的新生猪胰岛细胞与人淋巴细胞共孵育,通过ELISA法检测培养基中IL-2,IFN-γ,TNF-α细胞因子水平的变化。并观察转染组与对照组葡萄糖刺激后的胰岛素释放反应。结果:转染组NIPs可检测到hCTLA4-Ig基因及蛋白表达;与人淋巴细胞共孵育后IL-2,TNF-α,IFN-γ浓度明显低于对照组(P<0.01);且葡萄糖刺激胰岛素释放反应与对照组无明显差异(P>0.05)。结论:AAV-CTLA4-Ig体外转染的胰岛细胞能够表达具有生物学活性的hCTLA4-Ig,可抑制T细胞激活,而胰岛细胞生理功能不受影响。Objective To evaluate the expression of hCTLA4-Ig and their biological function in newborn porcine islets （NPIs） transfected with AAV-hCTLA4-Ig. Methods Cultured NPIs were transfected with AAV-hCTLA4-Ig. The expression of CTLA4-Ig in these NPIs was assayed by RT- PCR and immunocytochemistry. The levels of IL-2, IFN-γ, and TNF-α in the culture medium were assayed by ELISA after these cells the co-cultured with human. The response of glucose-stimulated insulin secretion was observed in the transgene group and the control group. Results The expres- sions of CTLA4-Ig mRNA and protein were detected in the transgene group. The levels of cytokines were obviously lower in the transgene group than those in the control group （ P 〈 0. 01 ）. There was no significant difference in the response of glucose-stimulated insulin release between the transgene group and the control group （ P 〉 0.05 ）.Conclusion AAV mediated hCTLA4-Ig expression in NPIs could inhibit T lymphocyte to produce cytokines, while the endocrine functions of the NPIs were not significantly affected.

关 键 词：CTLA4-IG 基因转染 新生猪胰岛细胞 

分 类 号：R657.5[医药卫生—外科学]