肺癌细胞抑制CD4+T细胞 IFN-γ基因表达的机制研究
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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)


The mechanism of downregulation of IFN-γ expression in CD4+T cells by lung cancer cells
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    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-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 = 0.010 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-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% vs 70.9%)-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-

    Abstract:

    Objective:To investigate whether lung cancer cells can directly influence the methylation of IFN-γ gene promoter in CD4+T cells. Methods:The plasma level of IFN-γ 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-γ 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-γ 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-γ promoter in CD4+ T cells of the experimental group was 85.4%(control,70.9%). Conclusion:Lung cancer cell can induce the hypermethylation of IFN-γ gene promoter,which downregulates the expression of the IFN-γ gene,and it may play an important role on the immunosuppression of lung cancer patients.

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秦雪君,潘世扬,王 芳,彭 蘡,徐 娟,韩 月,孙瑞红,张丽霞,王 宏,谢而付,高 丽,庞智睿.肺癌细胞抑制CD4+T细胞 IFN-γ基因表达的机制研究[J].南京医科大学学报(自然科学版),2012,(5):659-663

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  • 收稿日期:2011-12-22
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