RNA干扰白细胞抗原-G增强自然杀伤细胞对肝癌的免疫监视

RNA interference targeting human leukocyte antigen-G enhanced immune surveillance mediated by the natural kill cell on hepatocellular carcinoma

目的构建表达人类白细胞抗原-G（HLA—G）基凶shRNA载体，探讨其对自然杀伤细胞（NK）细胞杀伤效应的影响。方法构建4个HLA—G—shRNA真核表达质粒，转染至Huh7细胞。检测HLA—GmRNA及蛋白质水平的表达。将NK-92MI细胞与靶细胞共同培养，以LDH释放法观察不同效靶比时NK-92MI细胞对靶细胞的杀伤效应。结果经酶切及测序鉴定证实，插入序列与设计的序列相符。RealTime—PCR和Western-blot结果均表明重组载体抑制了Huh7细胞HLA-G基因的表达。NK-92MI细胞对转染HLA—G的shRNA的Huh7细胞杀伤作用明显升高（P〈0．01）。封闭NK-92MI细胞ILT2受体后，杀伤作用降低（P〈0．01）。结论HLA—GshRNA可以增强NK细胞对肝癌细胞的杀伤效应。

Objective To construct the expression vector of HLA-G-shRNA, and investigate the effect of HLA-G shRNA on the protection of Huh7 ce 11 lines from NK cell lysis. Methods Four HLA-G shRNA plasmids were constructed and transfected into Huh7 cell lines, and the levels of mRNA and protein of HLA-G were detected by real-time PCR and Western blot respectively. The cytotoxicity of NK-92MI cells against the transfected ceils was analyzed by LDH releasing assay. Results The gel electrophoresis and sequencing showed that the inserted sequence was identical to the one we designed, and no aberrations such as mutation, deletion or insertion occurred. The expression of HLA-G confirmed by real time-PCR and Western-blot was significantly down-regulated. Huh7 cell lines transfected with HLA-G shRNA showed higher lytic activity. After ILT2 receptor was blocked,lyric activity of NK-92MI cells was decreased. Conclusion HLA-G shRNA can increase NK cytolysis against Huh7 cells.