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目的探讨人胚肺成纤维细胞复制性衰老及过氧化氢诱导的早衰过程中Foxa2的表达改变及其启动子区CpG岛的甲基化水平变化。方法按传代情况将人胚肺成纤维细胞分为年轻细胞[第22代细胞,即22 PDL(populationdoubling levels,群体倍增水平)]组、中年细胞(35PDL)组、复制性衰老细胞(49PDL)组和氧化应激诱导的早衰细胞(premature senescence,PS)组。用荧光定量PCR方法检测人胚肺成纤维细胞衰老过程中Foxa2的mRNA表达改变,应用甲基化特异性PCR(MSP)检测启动子区-777～-478 bp甲基化的变化情况,并用亚硫酸氢盐修饰基因组结合克隆测序检测启动子区域CpG岛的甲基化水平。结果与年轻细胞组比较,中年细胞组人胚肺成纤维细胞Foxa2 mRNA的表达水平无明显变化;而复制性衰老细胞组和早衰细胞组人胚肺成纤维细胞Foxa2的mRNA表达水平均显著降低,差异有统计学意义(P<0.05)。早衰细胞组的启动子区具有一定的甲基化水平;年轻细胞组、复制性衰老细胞组及早衰细胞组Foxa2启动子区CpG岛的甲基化水平分别为5.7%,17.1%和43.6%。结论人胚肺成纤维细胞衰老过程中Foxa2的mRNA表达水平逐渐降低,其启动子区CpG岛甲基化水平逐渐升高,参与其表达调控。
Objective To investigate the changes of Foxa2 expression in human embryonic lung fibroblasts and hydrogen peroxide-induced premature senility and the methylation level of CpG island in human lung fibroblasts. Methods Human embryonic lung fibroblasts were divided into young cells [22nd generation cell population (population doubling levels)], middle-aged cell group (35PDL), replicative senescent cells (49PDL) Group and oxidative stress-induced premature senescence (PS) group. The mRNA expression of Foxa2 in senescent human embryonic lung fibroblasts was detected by real-time PCR. The methylation-specific PCR (MSP) was used to detect the methylation of -777-478 bp in the promoter region, The bisulfate modified genome was cloned and sequenced to detect the methylation level of CpG island in the promoter region. Results Compared with the young group, the expression of Foxa2 mRNA in the human embryo lung fibroblasts did not change significantly in the middle-aged group, while the expression of Foxa2 mRNA in the human embryonic lung fibroblasts was significantly decreased , The difference was statistically significant (P <0.05). The methylation level of promoter region of premature senescent cell group was 5.7%, 17.1% and 43.6%, respectively. The methylation levels of CpG island in Foxa2 promoter region of young cell group, replicating senescent cell group and premature senescent cell group were respectively 5.7%, 17.1% and 43.6%. Conclusion The mRNA expression of Foxa2 in human embryonic lung fibroblasts decreases gradually during the senescence, and the methylation level of CpG island in promoter region gradually increases, which is involved in the regulation of Foxa2 mRNA expression.