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目的建立一种准确、稳定的自然杀伤细胞(NK)活性检测方法。方法通过电穿孔法将含增强型绿色荧光蛋白(EGFP)的质粒转入K562细胞,经G418筛选后进行克隆,获得EGFP阳性表达细胞株;采用流式细胞仪和乳酸脱氢酶法检测10名健康人外周血NK细胞活性。结果通过基因转染的方法得到1株稳定表达EGFP的K562细胞,转染EGFP对细胞生长无影响,10名健康人外周血NK细胞对转染EGFP的细胞及未转染细胞的杀伤活性无差异(P>0.05);表达EGFP的K562细胞在效靶比为40∶1时,用流式细胞仪检测培养2和4h时的NK细胞杀伤率分别为(21.5±1.3)%和(23.7±1.5)%,二者无差异(P>0.05);而用乳酸脱氢酶法测定培养4h的NK细胞对K562和EGFP-K562的杀伤率[分别为(23.6±2.3)%和(23.7±2.3)%]均高于2h的杀伤率[(18.0±1.1)%和(18.2±1.3)%,P<0.05]。结论成功建立了表达EGFP的K562细胞株,将该细胞株作为靶细胞用于流式细胞术检测NK细胞活性时,无需预先染色和标记靶细胞,且操作简便、省时、稳定、可靠。
Objective To establish an accurate and stable method for the detection of natural killer (NK) activity. Methods The plasmid containing enhanced green fluorescent protein (EGFP) was transfected into K562 cells by electroporation, then screened by G418 and cloned to obtain EGFP positive cell lines. The expression of EGFP was detected by flow cytometry and lactate dehydrogenase Healthy human peripheral blood NK cell activity. RESULTS: One strain of K562 cells stably expressing EGFP was obtained by gene transfection. Transfection of EGFP did not affect the cell growth. There was no difference in the killing activity of NK cells transfected with EGFP and the untransfected cells among 10 healthy human NK cells (21.5 ± 1.3)% and (23.7 ± 1.5)%, respectively (P> 0.05). The NK cell killing rates of K562 cells expressing EGFP at the target ratio of 40:1 were (21.5 ± 1.3)% and ), Respectively. There was no significant difference between the two groups (P> 0.05); while killing rate of K562 and EGFP-K562 by NK cells cultured for 4h by lactate dehydrogenase [(23.6 ± 2.3)% and (23.7 ± 2.3) %] Were higher than 2h kill rate [(18.0 ± 1.1)% and (18.2 ± 1.3)%, P <0.05]. Conclusion The K562 cell line expressing EGFP was established successfully. When this cell line was used as a target cell for the detection of NK cell activity by flow cytometry, it was unnecessary to pre-stain and label the target cells, and its operation was simple, time-saving, stable and reliable.