新生猪胰岛细胞的HLA-G_1基因转染  

HLA-G_1 gene transfer to neonatal porcine islet cells mediated by genejammer

作　　者：曹荣华[1] 韩军艳[2] 吴雄文[2] 王树森[1] 陈栋[1] 祁洪刚[1] 夏振雄[1] 陈实[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院器官移植研究所,武汉430030 [2]同济医学院免疫学教研室

出　　处：《中华器官移植杂志》2005年第4期233-235,共3页Chinese Journal of Organ Transplantation

摘　　要：目的探讨HLA-G1基因转染新生猪胰岛细胞的可行性,检测其表达和功能状况。方法体外分离纯化新生猪胰岛细胞,脂质体载体转染HLA-G1基因。免疫荧光法检测HLA-G1基因在胰岛细胞上的表达状况;51Cr释放实验分析其对人NK细胞细胞毒的抑制作用。结果新生猪胰岛细胞HLA-G1基因表达率大约为50%,体外转染24-48 h达高峰;混合淋巴细胞培养(胰岛细胞+ NK细胞)后,空质粒转染组的胰岛细胞溶解率为71.57%,而转染组胰岛细胞溶解率为53.5%。结论利用脂质体Genejammer转染体外培养的新生猪胰岛细胞,可以获得靶基因的瞬时高效表达; HLA-G1基因转染可以有效抑制NK细胞对异种胰岛的细胞毒作用。Objective To study HLA-G1 gene transfection and function in the neonatal porcine islets (NPIs). Methods Pancreata were obtained from 3-5 day-old pigs and digested with collage-nase V. The neonatal porcine islets were cultured in vitro for 5 days, then the pcDNA3-HLA-G1 plas-mids were transferred to NPIs by vector of Genejammer. Surface expression of HLA-G1 was detected by indirect immunofluorescence. The expression of HLA-G1 was detected by immunofluorescence after 24 h and 48 h respectively. pcDNA3 transfection was performed as negative control. NK cells mediated NPIs lysis was evaluated by 51Cr release assays. Results After 5 days of culture in vitro, NPIs grew up vigorously. The peak time of pcDNA3-HLA-G1 transfer to NPIs was 24 h, and the transfection efficiency reached to about 53 % while DNA/liposome/cell complex was incubated for 48 h. Later, necrosis occurred in 10 %-15 % NPIs with the transfection efficiency being about 50 %. 51Cr release assays revealed that the lysis rate of pancreatic cells by NK cells was 71. 57 % in control group, while that in the pcDNA3-HLA-G1 transfection group was 53. 5 %. Conclusions Transfection of in vitro culture neonatal porcine islet cells by Genejammer can achieve transient expression of HLA-G1. Transfection of HLA-G1 cDNA into NPIs can effectively inhibit the cytoxicity of NK cells to pancreatic islet cells.

关 键 词：猪胰岛细胞 基因转染 新生 HLA-G1基因 混合淋巴细胞培养 脂质体载体 免疫荧光法 人NK细胞 细胞毒作用 功能状况 分离纯化 表达状况 抑制作用 实验分析 体外转染 体外培养 高效表达 异种胰岛 溶解率 表达率 靶基因 检测 

分 类 号：R587.1[医药卫生—内分泌]