摘要
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5 d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-γmRNA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=0.010 7)。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],6 h对照组CD4+T细胞IFN-γmRNA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基因表达下调,可能对肺癌患者的免疫抑制起重要作用。
Objective：To investigate whether lung cancer cells can directly influence the methylation of IFN-γ gene promoter in CD4＋T ceils. Methods：The plasma level of IFN-7 was determined by ELISA in the lung cancer patient group（n = 30） and healthy control group（n = 30）. CD4＋T cells were isolated by CD4-positive isolation kit from peripheral blood of lung cancer patients （n = 8） and healthy controls （n = 8）,and genomic DNA was extracted using QIAamp Mini Kit and bisulfite treated. IFN-γ gene promoter methylation was analyzed with methylation 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-A1 as the experimental group and CD4＋ T cells cultured as the control group. After culturing for 5 d,CD4＋ T cells were collected to analyze IFN-7 gene promoter methylation using methylation specific sequencing method as described above. Meanwhile,CD4＋ T cells were stimulated by anti-CD3 and anti-CD28 antibodies for 6 and 24 h. The IFN-γ/of supernatant was detected by ELISA 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 hypermethylation status of IFN-γ promoter in CD4＋ T cells of lung cancer patients was 84.6% （control,68.6%）（P 〈 0.001）. The concentration of plasma IFN-γ was negatively correlated with the percentage of methylation at the IFN-γ promoter in the patient group（r = -0.850 3,P = 0.010 7）. In Transwell culturing system,after stimulation for 6 and 24 h, the expression of IFN-3, 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
出处
《南京医科大学学报（自然科学版）》
CAS
CSCD
北大核心
2012年第5期659-663,共5页
Journal of Nanjing Medical University(Natural Sciences)
基金
国家自然科学基金(30972821
30901344
30901262)
江苏省实验诊断学重点实验室基金(XK201114)
