携带TGF-βⅡ型受体及NKG2D融合基因的NK-92细胞生物学特性研究  

Biological function of NK-92 cells carrying TGF-β receptor Ⅱ and NKG2D fusion gene

作　　者：王仲娟[1] 林丹丹[1] 张胤晟[1] 赵李祥[1] 刘海燕[1] 

机构地区：[1]苏州大学生物医学研究院肿瘤与细胞分子免疫实验室,215123

出　　处：《免疫学杂志》2013年第3期206-210,共5页Immunological Journal

摘　　要：目的构建携带TGF-βreceptorⅡ(TGF-βRⅡ)胞外段跨膜区及NKG2D胞内段融合基因的NK-92细胞,并检测其生物学特性。方法通过RT-PCR从人外周血淋巴细胞中扩增出TGF-βreceptorⅡ胞外段跨膜区及NKG2D胞内段基因;利用PCR的方法将2段基因融合,并构建含有融合基因的慢病毒穿梭质粒;将该质粒与包装质粒共转染293T细胞,获得慢病毒颗粒并感染NK-92细胞系,筛选出稳定转染的NK-92细胞系;采用流式细胞术检测目的基因在转染后的NK-92细胞系中的表达,用CCK-8的方法检测24 h和48 h时NK-92-TN的存活能力,同时用Transwell的方法检测NK-92-TN的迁移能力。结果测序表明,成功获得了TGF-βRⅡ胞外段跨膜区与NKG2D胞内段的融合基因;获得了重组慢病毒;通过筛选得到了可稳定表达融合基因的NK-92细胞系,同时NK-92-TN的存活能力和迁移能力要明显优于对照组。结论成功建立表达TN的NK-92细胞系,并且发现NK-92-TN的存活能力和迁移能力明显优于对照组,从而为NK-92-TN细胞的体内生物学功能的研究工作奠定了基础。This study designed to construct NK-92 stable cell lines carrying a fusion gene which consists of the extracellular and transmembrane domain of human TGF-β receptor Ⅱ and the intracellular domain of NKG2D（TN）, and to investigate the characterization of NK-92-TN. The fusion gene was obtained by PCR, and subsequently cloned to lentivirus shuttle plasmid. Recombinant lentivirus expressing the fusion gene was obtained through co-transfection of the lentivirus shuttle plasmid and a packaging plasmid into 293T cells. NK-92 cells were then infected with the recombinant virus, and the cells stably expressing the fusion gene were sorted by Arial-Ⅲ. CCK-8assay revealed that the viability of NK-92-TN was higher than that of the NK-92-venus control （P 〈 0.05）. Transwell results also demonstrated the migration ability of NK-92-TN was better than the NK-92-venus control.Taken together, our results suggest that NK-92-TN might be a new strategy for cancer therapy.

关 键 词：TGF-βⅡ型受体 NKG2D NK-92 存活 迁移 

分 类 号：R392.1[医药卫生—免疫学]