ADAM17下调影响NK细胞对非小细胞肺癌杀伤作用的实验研究

Effect of the down-regulation of ADAM17 on the killing effect of NK cells in non-small cell lung cancer

目的探讨非小细胞肺癌(NSCLC)中解聚素-金属蛋白酶17(ADAM17)下调对自然杀伤(NK)细胞杀伤作用的影响。方法体外培养人NSCLC A549细胞株,分为正常对照组(未转染)、阴性对照组(转染nonsense siRNA)和ADAM17下调组(转染ADAM17 siRNA)。采用实时荧光定量聚合酶链反应(qRT-PCR)检测各组A549细胞ADAM17 m RNA表达,Western blotting检测各组A549细胞ADAM17蛋白表达。制备NK细胞,加入各组A549细胞中,检测NK细胞杀伤活性,Western blotting检测各组细胞中γ干扰素(IFN-γ)、颗粒酶B(GraB)、穿孔素(PFP)蛋白表达。结果各组细胞ADAM17 mRNA、蛋白相对表达量比较,差异有统计学意义(P<0.05),正常对照组与阴性对照组细胞ADAM17 mRNA、蛋白相对表达量比较,差异无统计学意义(P>0.05),ADAM17下调组较正常对照组、阴性对照组降低(P<0.05)。各组NK细胞对A549细胞的杀伤活性比较,差异有统计学意义(P<0.05),正常对照组与阴性对照组NK细胞对A549细胞的杀伤活性比较,差异无统计学意义(P>0.05),ADAM17下调组较正常对照组、阴性对照组升高(P<0.05)。各组细胞中IFN-γ、GraB、PFP蛋白相对表达量比较,差异有统计学意义(P<0.05),正常对照组与阴性对照组细胞中IFN-γ、GraB、PFP蛋白相对表达量比较,差异无统计学意义(P>0.05),ADAM17下调组较正常对照组、阴性对照组升高(P<0.05)。结论NSCLC A549细胞株中ADAM17下调能增强NK细胞的杀伤作用。

Objective To investigate the effect of the down-regulation of A disintegrin and metalloproteinase17(ADAM17)on the killing effect of natural killer(NK)cells in non-small cell lung cancer(NSCLC).Methods Human NSCLC A549 cells were cultured in vitro and divided into normal control group(without transfection),negative control group(transfected with nonsense siRNA)and ADAM17 down-regulation group(transfected with ADAM17 siRNA).The m RNA level of ADAM17 in A549 cells was detected via quantitative real-time polymerase chain reaction(qRT-PCR),while the protein level of ADAM17 was determined via Western blotting(WB).NK cells were prepared and co-cultured with A549 cells of each group.The killing activity of NK cells was measured,and the protein levels of interferon-γ(IFN-γ),granzyme B(GraB)and perforin(PFP)were also detected via WB.Results The mRNA and protein levels of ADAM17,the protein levels of IFN-γ,GraB and PFP,and the killing activity of NK cells against A549 cells were different among the groups as a whole(P<0.05).Specifically,there were no significant differences in the m RNA and protein levels of ADAM17,the protein levels of IFN-γ,GraB and PFP,or the killing activity of NK cells against A549 cells between normal control group and negative control group(P>0.05),while the mRNA and protein levels of ADAM17,the protein levels of IFN-γ,GraB and PFP,and the killing activity of NK cells against A549 cells were lower in the ADAM17 down-regulation group compared with those in the normal control group and negative control group(P<0.05).Conclusions The down-regulation of ADAM17 in NSCLC A549 cells can enhance the killing effect of NK cells.