NK细胞中过表达Livin的实验研究

目的:将携带Livin的质粒pIRES2-EGFP-Livin进行扩增,转染自然杀伤细胞(NK)及胃癌细胞株SGC-7901,并检测其在NK及胃癌细胞株SGC-7901中的表达。方法:将携带Livin基因的质粒p IRES2-EGFP-Livin进行扩增,鉴定质粒纯度与浓度;从健康人外周血中获得NK细胞,应用HP转染试剂将质粒pIRES2-EGFP-Livin转染体外培养的NK及胃癌细胞,对比分析NK及胃癌细胞株SGC-7901中基因转染效率及目的基因的表达情况。结果:用无血清培养基在体外成功的扩增大量的NK细胞;质粒提取试剂盒抽提得到大量无内毒素的质粒,质粒DNA基因序列并未发生突变,浓度和纯度较高。胃癌细胞株SGC-7901中观察到明显的质粒pIRES2-EGFP-Livin绿色荧光表达;而NK中未观察到绿色荧光表达。结论:质粒pIRES2-EGFP-Livin能使Lvin蛋白表达于胃癌细胞株SGC-7901中,而在NK中未表达。

Overexpression of Livin Gene in Natural Killer Cells

Objective:To amplify the plasmid pIRES2-EGFP-Livin encoding functional human Livin gene to transfect naturalkiller cell (NK) and gastric carcinoma cell line SGC-7901, and test the expression in NK and gastric carcinoma cell line SGC-7901.Methods:The plasmid pIRES2-EGFP-Livin encoding functional human livin gene was amplified, the concentration and the purity ofplasmid pIRES2-EGFP-Livin were identified. NK from peripheral blood of healthy people were acquired And the plasmid pIRES2-EGFP-Livin was transfected into NK and gastric carcinoma cell line SGC-7901 using HP transfection reagent in vitro. The transfectionefficiency and the expression of human livin in NK and gastric carcinoma cell line SGC-7901 were compared.Results:A large quantityof NK cells were successfully amplified by serum-free medium in vitro; amounts of no endotoxin plasmid were acquired from PlasmidMidi Kit; plasmid had no mutations with high concentration and purity. The green bands of EGFP expression could be clearly observed inpIRES2-EGFP-Livin-transfected gastric carcinoma cell line SGC-7901, while could not be observed in NK.Conclusion:Effectiveexpressions of Livin protein had been verified in gastric carcinoma cell line SGC-7901 transfected by Plasmid pIRES2-EGFP-Livin butnot in NK.