T细胞人类白细胞抗原-G过表达对T细胞共培养胃癌MGC-803细胞的免疫杀伤作用

Immune killing effect of HLA-G over expression on co-cultured gastric cancer MGC-803 cells

目的研究T细胞人类白细胞抗原-G(HLA-G)过表达后对共培养胃癌MGC-803细胞的免疫杀伤作用。方法处死各组小鼠后分离淋巴细胞,通过免疫磁珠纯化T淋巴细胞,其纯度用流式细胞仪测定。将转染试剂、NC质粒、HLA-G重组载体分别转染至T淋巴细胞分为对照(Control)组、NC转染组、HLA-G转染组,细胞转染效率用荧光显微镜和流式细胞仪测定。以蛋白质印迹(WB)法测定各组细胞中HLA-G蛋白表达水平;以酶联免疫吸附(ELISA)法测定各组细胞上清液中γ-干扰素(IFN-γ)、白细胞介素-2(IL-2)、肿瘤坏死因子-α(TNF-α)表达水平;转染48 h后以CCK-8法检测HLA-G转染组、NC转染组、Control组与胃癌MGC-803细胞共培养时肿瘤免疫杀伤效率。结果HLA-G转染组细胞中HLA-G蛋白表达(1.08±0.09)明显高于Control组(0.18±0.04)和NC转染组(0.17±0.05,P<0.05)。与Control组、NC转染组相比,HLA-G转染组细胞中IFN-γ、IL-2、TNF-α水平均明显降低(均P<0.05)。效靶比为10∶1、20∶1、40∶1时,与Control组、NC转染组相比,HLA-G转染组胃癌细胞免疫杀伤率均显著降低(P<0.05)。结论T细胞过表达HLA-G后可减弱对胃癌细胞MGC-803体外免疫作用,当效靶比为40∶1时,T细胞对胃癌细胞MGC-803免疫杀伤作用最显著。

Objective To explore the immune killing effect of human leukocyte antigen-G(HLA-G)overexpression on T cell co-cultured gastric cancer MGC-803 cells.Methods After the mice of each group were killed,lymphocytes were collected from the spleen,T cells were purified by immunomagnetic beads,the purity of T lymphocyte was detected by flow cytometry.The T lymphocytes was transfected by transfection reagent,NC plasmid and HLA-G recombinant vector were divided into control group,NC transfection group,HLA-G transfection group.Flow cytometry and fluorescence microscope were used to detect cell transfection efficiency.Western blotting(WB)was used to detect the expression of HLA-G protein in cells;the expressions of interferon-γ(IFN-γ),interleukin-2(IL-2)and tumor necrosis factor-α(TNF-α)were detected by enzyme linked immunosorbent assay(ELISA);CCK-8 assay was used to detect the tumor immune killing efficiencies of HLA-G transfection group,NC transfection group control group were co-cultured with gastric cancer MGC-803 cells after 48 h of transfection.Results The expression of HLA-G protein(1.08±0.09)in HLA-G transfection group was significantly higher than those in control group(0.18±0.04)and NC group(0.17±0.05,P<0.05).The levels of IFN-γ,IL-2,TNF-αin HLA-G transfection group were significantly lower than those in control group and NC group(P<0.05).Compared with control group and NC transfection group,the mmune killing rate of gastric cancer cells in HLA-G transfection group was significantly lower when the target-effect ratio were 10∶1,20∶1 and 40∶1(P<0.05).Conclusion Overexpression of HLA-G in T cells attenuates the immune effect of MGC-803 on gastric cancer cells in vitro,when the target-to-effect ratio was 40∶1,T cells had the most significant immune killing effect on gastric cancer cell line MGC-803.