肺癌细胞抑制CD4+T细胞IFN-γ基因表达的机制研究

摘要

Objective;To investigate whether lung cancer cells can directly influence the methylation of IRN-7 gene promoter in CD4T cells. Methods;The plasma level of IEN-γ was determined by ELISA in the lung cancer patient group(n = 30) and healthy control group(n = 30). CD4T cells were isolated by CD4-positive isolation kil from peripheral blood of lung cancer patients (n = 8) and healthy controls (n = 8),and genomic DNA was extracted using Q1Aamp Mini Kit and bisulfite treated. IFN-γ gene promoter methylalion was analyzed with methylalinn specific sequencing method and the result was analyzed by bioinformatics software;A Transwell culturing system was also established(n = 6). CD4+ T cells of healthy volunteers were co-cultured with SPC-AI as the experimental group and CD4+ T cells cultured as the control group. After culturing for 5 d,Cl)4+ T cells were collected to analyze IFN-7 gene promoter methylution using methvlation specific sequencing method as described above. Meanwhile, CD4+ T cells were stimulated by anti-CI)3 and nnti-CI)2K antilxidies for 6 and 24 h. The IFN-7 of supernatant was delected by EIISA and RT-PCR was used to determine the mRNA transcript levels of IFN-γ. Results; The level of plasma IFN-γ was significantly lower in lung cancer patients(69.30 ± 38.56 pg/ml vs 92.62 ± 34.75 pg/ml,P= 0.017). The hypermelhylation status of IFN-7 promoter in CD4+ T cells of lung cancer patients wan 84.6%(controll,68.6%)(P < 0.001). The concentration of plasma IFN-7 was negatively correlated with the percentage of methylalion at the IFN-γ promoter in the patient group(r = -0.850 3,P = 0.010 7). In Transwell culluring system,after stimulation for 6 and 24 h.the expression of IFN-7 in the experimental group was significantly lower than that of the control group [6 h:( 14.53 ± 7.12) pg/ml vs (36.14 ± 23.51) pg/ml,24 h:(7.81 ± 4.02) pg/ml vs (24.85 ± 15.58) pg/ml]. The mRNA transcript levels of IFN-γ of the control groups were increased 2.37 fold after stimulation for 6 h. The hypermethylation status of the IFN-7 promoter in CD4+ T cells of the experimental group was 85.4%(control,70.9%). Conclusion:Lung cancer cell can induce the hyperme-thylation of IFN-7 gene promoter, which dcwnregulates the expression of the IFN-7 gene,and it may play an important role on the im-munosuppression of lung cancer patients.%目的:研究肺癌细胞能否直接影响CD4+T细胞干扰素-γ(IFN-γ)基因启动子甲基化水平.方法:ELISA法检测肺癌组(n =30)及健康对照组(n=30)血浆IFN-γ水平；免疫磁珠分选两组外周血CD4+T细胞(n=8),提取DNA后经亚硫酸氢盐修饰,PCR扩增IFN-γ基因启动子进行TA克隆测序,测序结果采用生物信息学软件进行分析；建立健康人CD4+T细胞与 肺腺癌细胞株SPC-A1体外Transwell共培养体系(n=6),并设健康人CD4+T细胞单独培养为对照组,培养5d后分别收集CD4+T细胞.CD4+T细胞按上述法进行TA克隆测序.同时CD4+T细胞经anti-CD3、anti-CD28刺激6、24 h,ELISA法检测两组上清IFN-γ表达水平,RT-PCR检测CD4+T细胞IFN-γ mRNA表达水平.结果:肺癌组血浆IFN-γ水平显著低于健康对照组[(69.30±38.56) pg/ml vs (92.62±34.75) pg/ml,P=0.017]；肺癌组CD4+T细胞IFN-γ基因启动子甲基化水平显著高于健康对照组(84.6％ vs 68.6％,P＜0.001)；肺癌患者血浆IFN-γ水平与其基因启动子甲基化率呈负相关(r=-0.850 3,P=0.0107).体外Transwell共培养实验中,与对照组相比,实验组CD4+T细胞anti-CD3、anti-CD28刺激6、24 h,IFN-γ表达水平显著下降[6 h:(14.53±7.12) pg/ml vs (36.14±23.51) pg/ml,24 h:(7.81±4.02) pg/ml vs (24.85±15.58) pg/ml],6h 对照组CD4+T细胞IFN-γ mRNA表达水平为实验组的2.37倍.实验组CD4+T细胞IFN-γ基因启动子甲基化水平显著高于对照组(85.4％ vs70.9％).结论:肺癌细胞可诱导CD4+T细胞IFN-γ基因启动子发生高甲基化,进而导致IFN-γ基因表达下凋,可能对肺癌患者的免疫抑制起重要作用.