肝癌Hep3B细胞HLA-G过表达对NK细胞体外杀伤作用的影响

Effect of overexpression of human leukocyte antigen-G in hepatocellular carcinoma Hep3B cells on killing activity of NK cells in vitro

目的:探讨肝癌Hep3B细胞白细胞抗原-G(HLA-G)过表达对NK细胞体外杀伤作用的影响。方法:实验分为正常对照组(无干预措施,Hep3B组)、空载体对照组(转染空质粒,Hep3B-GFP组)和实验组(转染pEGFP-C1-HLA-G质粒,Hep3B-HLA-G组)。构建HLA-G真核表达质粒,转染至Hep3B细胞,经G418筛选,建立稳定过表达HLA-G的肝癌Hep3B细胞。Real-time PCR及Western blotting检测肝癌Hep3B细胞HLA-G mRNA及蛋白质水平的表达。分析肝癌Hep3B细胞HLA-G过表达及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:经酶切及测序鉴定证实,插入序列与设计的序列相符。Real-time PCR和Western blotting结果均表明,转染重组质粒的Hep3B细胞HLA-G过表达(P<0.01)。NK细胞对转染pEGFP-C1-HLA-G的Hep3B细胞杀伤作用明显降低(P<0.01),HLA-ABC单抗封闭靶细胞相应位点后,NK细胞对Hep3B、Hep3B-GFP和Hep3B HLA-G细胞的杀伤均增加(P<0.01)。结论:pEGFP-C1-HLA-G表达载体可有效增加HLA-G表达,HLA-G过表达可以削弱NK细胞对肝癌细胞的杀伤效应。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 of up-regulation of human leukocyte antigen-G（HLA-G） expression in hepatocellular carcinoma Hep3B cells on the killing effect of NK cells in vitro.METHODS： The following groups were set up： normal control group（without intervention,Hep3B group）,empty vector control group（transfection of empty plasmid,Hep3B-GFP group）,and experimental group（transfection of plasmid pEGFP-C1-HLA-G,Hep3B-HLA-G group）.The pEGFP-C1-HLA-G plasmid was constructed and transfected into Hep3B cells.The mRNA and protein levels of HLA-G were detected by real-time PCR and Western blotting.The cytotoxicity of NK cells against the Hep3B cells that overexpressed HLA-G,and against the target cells that the action sites on the surface of the cells were blocked by HLA-ABC antibody were analyzed.RESULTS： The results of gel electrophoresis and sequencing showed that the inserted sequence was identical to the one we designed and had no aberrations such as mutation,deletion or insertion.After transfection of recombinant plasmid,overexpression of HLA-G in Hep3B cells was confirmed by real-time PCR and Western blotting（P0.01）.Hep3B cells transfected with pEGFP-C1-HLA-G showed lower cyyolytic activity.After the corresponding sites of the target cells were blocked by HLA-ABC antibody,the cytolytic activity of NK cells against Hep3B,Hep3B-GFP and Hep3B HLA-G cells was increased（P0.01）.CONCLUSION： The pEGFP-C1-HLA-G was constructed in vitro and HLA-G was overexpressed at mRNA and protein levels in Hep3B cells.Overexpression of HLA-G decreases NK cytolysis against Hep3B cells.Blockage of the sites on the surface of the target cells by HLA-ABC antibody universally enhances the killing effect of NK cells on the target cells.