肝癌Bel7402细胞HLA-E基因上调对NK细胞体外杀伤作用的影响

Influence on killing effect of NK cells in vitro by up-regulation of HLA-E expression in hepatocellular carcinoma Bel7402 cells

目的:探讨肝癌Bel7402细胞人类白细胞抗原-E(HLA-E)基因上调对NK细胞体外杀伤作用的影响。方法:体外构建Lentivirus/CMV/GFP-HLA-E慢病毒表达载体并转导肝癌Bel7402细胞,采用RT-PCR和Western blotting方法检测肝癌Bel7402细胞HLA-E基因mRNA水平和蛋白水平的表达,分析肝癌Bel7402细胞HLA-E基因上调及HLA-ABC抗体封闭靶细胞相应位点对NK细胞体外杀伤作用的影响。结果:Lentivirus/CMV/GFP-HLA-E重组慢病毒载体感染Bel7402细胞后不同时段(24h、48h、72h、96h)的HLA-E mRNA的表达明显上调,除在感染后24h(P<0.05)外,慢病毒过表达组在感染后48h、72h、96h与Bel7402组比较,显著差异(P<0.01)。蛋白水平的表达亦明显上调:24h、48h、72h、96h外源性/内源性HLA-E吸光度比值与12h比较显著差异(P<0.01)。未封闭的Bel7402组和Bel7402 LentiHLA-E组比较,NK细胞的杀瘤活性差异显著(P<0.05或P<0.01);封闭组和未封闭组比较,NK细胞的杀瘤活性差异显著(P<0.05);Bel7402(封闭组)和Bel7402 LentiHLA-E(封闭组)比较,NK细胞的杀瘤活性有显著差异(P<0.01)。结论:Lentivirus/CMV/GFP-HLA-E慢病毒表达载体能有效增加HLA-E的表达。NK细胞对HLA-E基因表达上调的Bel7402细胞的靶杀伤作用降低;抗HLA-ABC单抗封闭靶细胞相应位点后,NK细胞对靶细胞的杀伤能力普遍有所提高。

AIM ： To investigate the influence on the killing effect of NK cells in vitro by up - regulation of human leukocyte antigen- E （HLA- E） expression in bepatocellular carcinoma Be17402 cells. METHODS： The recombinant lentiviral vector （ Lentivirus/CMV/GFP - HLA - E） was constructed and transfected into hepatocellular carcinoma Be17402 cells. The HLA - E genc expression at mRNA and protein level was monitored by the methods of real - time RT - PCR and Western blotting. The influence on the killing effect of NK cells in vitro by up - regulation of HLA - E expression in hepatocellular carcinoma Be17402 cells and by HLA - ABC antibody blocking the site on the surface of target cells was analyzed. RESULTS： Real - time RT - PCR showed that there was a significant increase in HLA - E mRNA level in hepatocellular carcinoma Be17402 cells transfected with Lentivims/CMV/GFP - HLA - E at 24 h （ P 〈0. 05 ） and 48 h,72 h,96 h （P 〈0. 01 ） as compared to blank group. There was also a significant increase in exogenous/endogenous HLA - E proteins at 12 h, 24 h, 48 h, 72 h and 96 h by Western blotting （P〈0. 01）. Without HLA -ABC antibody blocking, there was a statistical difference for the killing effect of NK cells, comparing Bcl7402 Lenti HLA - E group with Be17402 group （ P 〈 0. 05 ）. Comparing HLA - ABC antibody blocking group with no HIA - ABC antibody blocking group, a statistical difference for the killing eBect of NK cells （ P 〈 0. 05） was observed. There was also a statistical difference for the killing effect of NK cells between Be17402 with blocking group and Be17402 Lenti HLA - E with blocking group （ P 〈 0. 05 ）. CONCLUSION： The vector of Lenfivirus/CMV/GFP - HLA - E has an active up - regulation effect in HLA - E mRNA level and HLA - E protein level. While up - regulation of HLA - E in target cells, the killing effect of NK cells on target cells is obviously weakened. Blockage of the sites on the surface of target cells by HLA - ABC antibody universally en- hances the killing effect of NK cells on the target cells.