体外扩增外周血NK细胞对不同肿瘤细胞系的细胞毒作用

Cytotoxic effect of peripheral blood NK cell proliferation in vitro on different tumor cell lines

目的探讨体外扩增的外周血NK细胞对不同肿瘤细胞系的细胞毒作用。方法人肝癌细胞株BEL-7402、HepG2、SMCC-7721,人肺癌细胞株A549和人宫颈癌细胞株Hela均为中山大学附属第三医院岭南医院生物治疗中心实验室保存并传代。10例健康志愿者外周血均采集自中山大学附属第三医院岭南医院,其中男5例,女5例;平均年龄(52±13)岁。所有受试者均签署知情同意书,符合医学伦理学规定。分离人外周血单核细胞,培养NK细胞,并采用流式细胞术鉴定。采用CCK-8法检测体外扩增的人外周血CD56+CD3-NK细胞对肿瘤细胞毒作用;观察不同波长、培养时间、靶细胞数和不同效靶比等条件下吸光度(A)值。结果人外周血培养14 d后,CD56+CD3-NK细胞占(61±24)%。靶细胞最佳吸收波长为450 nm,最佳测定时间1~4 h,最佳细胞数量(1~4)×10^(4)个/ml。NK细胞对不同肿瘤细胞系的细胞毒作用不同,其中在效靶比40∶1时,对BEL7402作用最强,其后依次为A549、SMMC7721、HepG2,作用最弱为Hela;在20∶1、10∶1时,对BEL7402作用最强,其后依次为SMMC7721、A549、HepG2,作用最弱为Hela。NK细胞对3株肝癌细胞的细胞毒作用也不同,其中对BEL7402作用最强,SMMC7721次之,HepG2最弱。NK细胞在效靶比40∶1时对BEL-7402、SMCC-7721、A549、Hela细胞的杀伤率明显高于10∶1时(LSD-t=2.10,2.37,2.26,5.61;P<0.05);NK细胞在效靶比40∶1时对A549细胞的杀伤率明显高于20∶1时(LSD-t=1.14,P<0.05)。结论效靶比40∶1时NK细胞对各肿瘤细胞系的细胞毒作用最强。NK细胞对不同肿瘤细胞系的细胞毒作用不同,对同一种肿瘤不同细胞株的细胞毒作用也不同。

Objective To investigate the cytotoxic effect of peripheral blood NK cell proliferation in vitro on different tumor cell lines.Methods Human hepatoma cell strains BEL-7402,HepG2 and SMCC-7721,human lung cancer cell strain A549 and human cervical cancer cell strain Hela were stored and passaged in the laboratory of Biological Treatment Center of Lingnan Hospital,the Third Affiliated Hospital of Sun Yat-sen University.The peripheral blood samples were collected from 10 healthy volunteers,5 male and 5 female,aged(52±13)years on average,from Lingnan Hospital,the Third Affiliated Hospital of Sun Yat-sen University.The informed consents of all subjects were obtained and the local ethical committee approval was received.Human peripheral blood mononuclear cells were isolated,NK cells were cultured and identified by flow cytometry.The cytotoxic effect of human peripheral blood CD56+CD3-NK cell proliferated in vitro on the tumor cells was evaluated by CCK-8 method.The absorbance(A)value was observed under different conditions of wavelengths,culture time,target cell counts and different effect to target ratio.Results After human peripheral blood was cultured for 14 d,CD56+CD3-NK cells accounted for(61±24)%.The optimal absorption wavelength of target cells was 450 nm,the optimal detection time was 1-4 h,and the optimal cell density was(1-4)×10^(4) cells/ml.NK cells exerted different cytotoxic effects on different tumor cell lines.At the effect to target ratio of 40∶1,NK cells had the highest effect on BEL7402,followed by A549,SMMC7721,HepG2 and Hela cell lines.At the effect to target ratio of 20∶1 and 10∶1,NK cells exerted the highest effect on BEL7402,followed by SMMC7721,A549,HepG2 and Hela cell lines.The cytotoxic effects of NK cells on 3 hepatoma cell strains also differed.The effect on BEL7402 was the highest,followed by SMMC7721 and HepG2 cell lines.At the effect to target ratio of 40∶1,the killing probability of NK cells on BEL-7402,SMCC-7721,A549 and Hela cells were significantly higher than those at 10∶1(LSD-t=2.10,2.37,2.26,5.61;P<0.05).The killing probability of NK cells on A549 cells was significantly higher when the effect to target ratio was 40∶1,compared with 20∶1(LSD-t=1.14,P<0.05).Conclusions NK cells exert the highest cytotoxic effect on the tumor cell lines at the effect to target ratio of 40∶1.NK cells have different cytotoxic effects on different tumor cell lines,and also exert varying cytotoxic effects on different cell strains of the same tumor cell line.