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目的构建表达人类白细胞抗原-G(HLA-G)基因shRNA载体,探讨其对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,瞬时转染至人肝癌Bel-7402细胞,检测HLA-G mRNA和蛋白水平的表达。将NK-92MI细胞(效应细胞)与转染后的Bel-7402细胞(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。RT-PCR和Western blot结果均表明重组载体抑制了Bel-7402细胞HLA-G基因的表达。NK-92MI细胞对转染HLA-GshRNA的Bel-7402细胞杀伤作用明显增强(P<0.01)。封闭NK-92MI细胞KIR2DL4受体后,杀伤作用减弱(P<0.01)。结论成功构建了HLA-G shRNA载体,下调HLA-G可以增强NK细胞对肝癌细胞的杀伤效应,HLA-G与NK细胞表面KIR2DL4受体结合后,向NK细胞传递抑制效应。
Objective To construct a shRNA vector expressing human leukocyte antigen-G (HLA-G) gene and investigate its effect on NK cell killing effect. Methods Four HLA-G-shRNA eukaryotic expression plasmids were constructed and transiently transfected into human hepatoma Bel-7402 cells to detect the expression of HLA-G mRNA and protein. NK-92MI cells (effector cells) were co-cultured with the transfected Bel-7402 cells (target cells), and the killing effect of NK-92MI cells on target cells was observed by LDH release assay. Results confirmed by restriction enzyme digestion and sequencing confirmed that the inserted sequence was consistent with the designed sequence. The results of RT-PCR and Western blot showed that the recombinant vector inhibited the expression of HLA-G gene in Bel-7402 cells. The killing effect of NK-92MI cells on Bel-7402 cells transfected with HLA-G shRNA was significantly enhanced (P <0.01). After blocking the KIR2DL4 receptor of NK-92MI cells, the killing effect was weakened (P <0.01). Conclusion The HLA-G shRNA vector was successfully constructed. Down-regulation of HLA-G can enhance the killing effect of NK cells on hepatocellular carcinoma cells. After binding to KIR2DL4 receptor on the surface of NK cells, HLA-G can transmit inhibitory effect to NK cells.