免疫效应细胞增殖与细胞毒性功能检测3种方法的比较

A comparative study on three detection methods of proliferation and cytotoxic function in immune effector cells

目的:对比研究用于检测免疫效应细胞增殖与细胞毒功能的方法。方法:分别采用CCK-8法、EdU标记法、CFSE标记法检测不同培养条件下NK92细胞增殖[组1:100 U/mL白细胞介素(interleukin,IL)-2;组2:100 U/mL IL-2+10 U/mL IL-15]。用乳酸脱氢酶(lactate dehydrogenase,LDH)释放法、活细胞染料双标流式法、荧光素酶法检测两组NK92对K562细胞的杀伤。结果:CCK-8法检测结果提示组2增殖能力强于组1,但差异无统计学意义(P>0.05),而EdU和CFSE标记法均提示两组间增殖能力有显著差异(P<0.05)。活细胞染料双标流式法与荧光素酶法均能够检测出不同效靶比下两组NK92细胞毒功能的差异(P<0.05),LDH释放法在低效靶比下未检测出两组细胞毒性的差异(P>0.05)。以双标流式法的检测值为标准参照,荧光素酶法和LDH释放法的检测值与其呈显著相关性(rS=0.979 4,P<0.001;rS=0.973 2,P<0.001)。结论:CCK-8法更侧重于检测代谢活性,EdU和CFSE标记法更适用于悬浮类免疫细胞的增殖检测。3种细胞毒功能检测具有一致性,活细胞染料双标流式法适合检测对悬浮类靶细胞的杀伤,荧光素酶法比LDH释放法更适用于检测对贴壁细胞的杀伤作用。

Objective:This study aims to systematically compare the methods for detecting the proliferation and cytotoxic function of immune effector cells. Methods:CCK-8 assay,EdU labeling assay and CFSE labeling assay were used to detect the proliferation of NK92 cells in vitro under different culture conditions(group 1:100 U/mL IL-2;group 2:100 U/mL IL-2+10 U/mL IL-15),and the killing effect of NK92 on K562 cells was detected by lactate dehydrogenase(LDH)release assay,double living cell dye labeling assay and luciferase assay. The principles,characteristics and effectiveness of the various methods were compared. Results:The results of CCK-8 assay showed that the proliferation ability of group 2 was stronger than that of group 1,but there was no significant difference between group 2 and group 1(P > 0.05),but both EdU and CFSE labeling assay showed that there were significant differences in proliferation ability between the two groups. Double living cell dye labeling flow assay and luciferase assay could detect the differences in cytotoxic function of NK92 between the two groups under different effector-target ratio(P < 0.05),but there was no difference in cytotoxicity between the two groups under low effector-target ratio detected by LDH release assay(P > 0.05). When double living cell dye labeling flow assay was as standard method,results of luciferase assay and LDH release assay were significantly correlated with the results of living cell dye labeling flow assay(rS< 0.979 4,P < 0.001;rS=0.973 2,P < 0.001). Conclusion:CCK-8 assay is more focused on the detection of metabolic activity,Ed U and CFSE labeling assay are more suitable for detecting the proliferation of suspended immune cells. The three kinds of cytotoxic function assay are consistent. Double living cell dye labeling assay is suitable for detecting the killing of suspended target cells,and luciferase assay is more suitable than LDH release method for the killing of adherent cells.