论文部分内容阅读
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。
OBJECTIVE: To amplify plasmid pIRES2-EGFP-Livin carrying Livin, and to transfect natural killer (NK) and gastric cancer cell line SGC-7901, and to detect its expression in NK and gastric cancer cell line SGC-7901. Methods: Plasmid pIRES2-EGFP-Livin carrying Livin gene was amplified to identify plasmid purity and concentration. NK cells were obtained from peripheral blood of healthy volunteers. Plasmid pIRES2-EGFP-Livin was transfected in vitro by HP transfection reagent Of NK and gastric cancer cells, comparative analysis of NK and gastric cancer cell line SGC-7901 gene transfection efficiency and the expression of the target gene. Results: A large number of NK cells were successfully expanded in serum-free medium in vitro. Plasmid extraction kit was used to extract a large number of endotoxin-free plasmids. The sequence of plasmid DNA did not mutate and its concentration and purity were high. Obvious green fluorescence expression of pIRES2-EGFP-Livin was observed in gastric cancer cell line SGC-7901, but no green fluorescence was observed in NK cells. CONCLUSION: The plasmid pIRES2-EGFP-Livin can induce Lvin protein expression in gastric cancer cell line SGC-7901, but not in NK cells.