IL-18/IL-18BP介导NK-92MI细胞杀伤GTKO猪内皮细胞的初步研究

Preliminary study of the role of IL-18/IL-18BP in mediating cytotoxic ability of NK-92MI cells against endothelial cells from GTKO porcine models

目的探讨白细胞介素(IL)-18/IL-18结合蛋白(BP)介导自然杀伤(NK)-92MI细胞杀伤α-1,3-半乳糖基转移酶(GGTA1)基因敲除(GTKO)猪内皮细胞的效应及可能机制。方法将NK-92MI细胞分为NK组、NK+IL-18组、NK+GTKO组、IL-18+NK+GTKO组和IL-18+IL-18BP+NK+GTKO组,采用实时定量逆转录聚合酶链反应(qRT-PCR)检测NK-92MI细胞中炎症相关基因的信使核糖核酸(mRNA)表达,乳酸脱氢酶(LDH)法检测NK-92MI细胞对GTKO猪内皮细胞的杀伤效应,脱氧核糖核酸末端转移酶介导的dUTP缺口末端标记(TUNEL)法检测GTKO猪内皮细胞的凋亡情况,蛋白质印迹法检测具有杀伤效应的蛋白和凋亡相关蛋白的表达。结果与NK组、NK+IL-18组以及NK+GTKO组比较,IL-18+NK+GTKO组NK-92MI细胞中干扰素(IFN)-γ、肿瘤坏死因子(TNF)-α、IL-8、IL-3、IL-6和粒细胞-巨噬细胞集落刺激因子(GM-CSF)的mRNA表达水平均升高,差异均有统计学意义(均为P<0.05)。与IL-18+NK+GTKO组比较,IL-18+IL-18BP+NK+GTKO组NK-92MI细胞中IFN-γ、TNF-α、IL-8、IL-3、IL-6和GM-CSF的mRNA表达水平均降低,差异均有统计学意义(均为P<0.05)。与NK+GTKO组相比,IL-18+NK+GTKO组NK-92MI细胞中穿孔素、颗粒酶B和IFN-γ蛋白表达水平均升高,NK-92MI细胞对GTKO猪内皮细胞的杀伤率增加,GTKO猪内皮细胞凋亡率增加,GTKO猪内皮细胞中B淋巴细胞瘤-2(Bcl-2)相关X蛋白(Bax)/Bcl-2和裂解半胱氨酸天冬氨酸蛋白酶(cleaved Caspase)-3/半胱氨酸天冬氨酸蛋白酶(Caspase)-3的比例均升高,差异均有统计学意义(均为P<0.05)。与IL-18+NK+GTKO组比较,IL-18+IL-18BP+NK+GTKO组穿孔素、颗粒酶B、IFN-γ蛋白表达水平均降低,NK-92MI细胞对GTKO猪内皮细胞的杀伤率降低,GTKO猪内皮细胞凋亡率下降,GTKO猪内皮细胞中Bax/Bcl-2和cleaved Caspase-3/Caspase-3的比例均下降,差异均有统计学意义(均为P<0.05)。结论IL-18BP可阻断IL-18诱导的NK-92MI细胞中炎症相关基因表达和NK-92MI细胞杀伤GTKO猪内皮细胞的效应。

Objective To evaluate the role and potential mechanism of interleukin(IL)-18/IL-18 binding protein(BP in mediating the killing effect of natural killer(NK)-92MI cells upon endothelial cells fromα-1,3-galactosyltransferase gene-knockout(GTKO)porcine models.Methods NK-92MI cells were divided into the NK,NK+IL-18,NK+GTKO,IL-18+NK+GTKO and IL-18+IL-18BP+NK+GTKO groups.The messenger ribonucleic acid(mRNA)levels of inflammationrelated genes in NK-92MI cells were detected by quantitative real-time reverse transcription polymerase chain reaction(qRT-PCR).The killing effect of NK-92MI cells on endothelial cells from GTKO porcine models was evaluated by lactate dehydrogenase(LDH)assay.The apoptosis of endothelial cells from GTKO porcine models was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling(TUNEL)assay.The expression levels of proteins with killing effect and apoptosis-related proteins were determined by Western blot.Results Compared with the NK,NK+IL-18and NK+GTKO groups,the expression levels of interferon(IFN)-γ,tumor necrosis factor(TNF)-α,IL-8,IL-3,IL-6and granulocyte-macrophage colony stimulating factor(GM-CSF)mRNA were up-regulated in NK-92MI cells in the IL-18+NK+GTKO group,and the differences were statistically significant(all P<0.05).Compared with the IL-18+NK+GTKO group,the expression levels of IFN-γ,TNF-α,IL-8,IL-3,IL-6 and GM-CSF mRNA were down-regulated in NK-92MI cells in the IL-18+IL-18BP+NK+GTKO group,and the differences were statistically significant(all P<0.05).Compared with the NK+GTKO group,the expression levels of perforin,granzyme B and IFN-γproteins in NK-92MI cells were up-regulated,the killing rate of NK-92MI cells against endothelial cells from GTKO porcine models was enhanced,the apoptosis rate of endothelial cells from GTKO porcine models was increased,and the ratios of B cell lymphoma-2(Bcl-2)-associated X protein(Bax)/Bcl-2 and cleaved Caspase-3/Caspase-3 in endothelial cells from GTKO porcine models were elevated in the IL-18+NK+GTKO group,and the differences were statistically significant(all P<0.05).Compared with the IL-18+NK+GTKO group,the expression levels of perforin,granzyme B and IFN-γproteins were down-regulated,the killing rate of NK-92MI cells against endothelial cells from GTKO porcine models was decreased,the apoptosis rate of endothelial cells from GTKO porcine models was decreased,and the ratios of Bax/Bcl-2 and cleaved Caspase-3/Caspase-3in endothelial cells from GTKO porcine models were declined in the IL-18+IL-18BP+NK+GTKO group,and the differences were statistically significant(all P<0.05).Conclusions IL-18BP may block the expression of inflammationrelated genes in NK-92MI cells induced by IL-18 and the killing effect of NK-92MI cells on endothelial cells from GTKO porcine models.