白细胞介素6启动子通过调控人端粒酶逆转录酶减轻细胞炎症和DNA损伤

doi:10.3877/cma.j.issn.1674-6880.2024.02.003

目的

探讨外源性白细胞介素6（IL-6）启动子调控外源性人端粒酶逆转录酶（hTERT）表达情况及其对细胞衰老的干预作用。

方法

将IL-6启动子与增强型绿色荧光蛋白（EGFP）（GV3-IL-EGFP）或携带EGFP标记的hTERT（GV3-IL-hT-EGFP）融合构成慢病毒载体，收集相应病毒颗粒，并用病毒颗粒转染第5代人皮肤成纤维细胞（HFF-1）。通过自然传代细胞构建复制性细胞衰老模型，将转染GV3-IL-EGFP重组病毒的HFF-1作为对照组，转染GV3-IL-hT-EGFP重组病毒的HFF-1作为基因治疗组。细胞接近完全衰老时（第19代和第25代）检测两组细胞hTERT蛋白表达水平，并绘制细胞群体倍增水平（PDL）曲线。在两组细胞生长速度出现差异时（第13代、第14代、第15代）检测紫外线辐射敏感蛋白（RAD51）和磷酸化H2A组蛋白家族成员X（γ-H2AX）蛋白表达水平。在第15代、第17代、第19代时检测两组细胞的IL-6和IL-1α蛋白表达水平。同时，对第13代、第14代、第15代细胞给予阿霉素100 nM处理48 h后，比较两组细胞RAD51和γ-H2AX的蛋白表达水平。

结果

对照组第19代、基因治疗组第19代及第25代hTERT蛋白表达水平比较，差异具有统计学意义（F = 941.781，P < 0.001）；与对照组第19代比较，基因治疗组细胞第19代和第25代hTERT蛋白表达水平均显著升高（P均< 0.001）。基因治疗组的第17代和第19代IL-6（t = 61.570，P < 0.001；t = 36.527，P < 0.001）和IL-1α（t = 11.984，P < 0.001；t = 18.622，P < 0.001）蛋白表达较对照组均显著下调。对照组细胞RAD51和γ-H2AX蛋白表达水平在第14代（t = 3.101，P = 0.036；t = 10.226，P = 0.001）和在第15代（t = 8.683，P = 0.001；t = 7.229，P = 0.002）较基因治疗组均显著升高。PDL曲线显示，在第19代时对照组细胞停止增殖并处于完全衰老状态，而基因治疗组细胞仍具有增殖能力（t = 6.856，P = 0.002）。同时经过阿霉素处理后，在第13代、第14代和第15代时，基因治疗组细胞RAD51（t = 23.479，P < 0.001；t = 9.619，P = 0.001；t = 14.971，P < 0.001）、γ-H2AX（t = 4.275，P = 0.013；t = 6.787，P = 0.002；t = 9.569，P = 0.001）较对照组均明显降低。

结论

经过基因治疗后的细胞，在复制性细胞衰老和阿霉素诱导细胞衰老的过程中，可以借助IL-6启动子调控hTERT的表达，减少DNA损伤积累，减轻炎症因子水平。

关键词: 白细胞介素6, 细胞衰老, 衰老相关分泌表型, 端粒酶逆转录酶, DNA损伤

Objective

To investigate the regulation of exogenous human telomerase reverse transcriptase (hTERT) expression by the exogenous interleukin-6 (IL-6) promoter and its effect on cell senescence.

Methods

The IL-6 promoter was fused with enhanced green fluorescent protein (EGFP) (GV3-IL-EGFP) or hTERT-EGFP (GV3-IL-hT-EGFP) to construct lentiviral vectors. The corresponding virus particles were collected and transfected into the fifth generation human foreskin fibroblasts-1 (HFF-1). The replicative cell senescence model was constructed by natural passage cells, and HFF-1 transfected with recombinant virus of GV3-IL-EGFP was used as the control group and HFF-1 transfected with recombinant virus of GV3-IL-hT-EGFP was used as the gene therapy group. The expression level of hTERT protein in cells of the two groups was detected when the cells were approaching complete senescence (19th and 25th generations), and the curve of cell population doubling level (PDL) was drawn. The expression levels of radiation sensitive protein 51 (RAD51) and gamma-H2A histone family member X (γ-H2AX) proteins were detected when the growth rates of cells in the two groups were different (13th, 14th and 15th generations). The expression levels of IL-6 and IL-1α proteins were detected at the 15th, 17th and 19th generations. At the same time, the protein expression levels of RAD51 and γ-H2AX were compared between the two groups after 100 nM doxorubicin treatment for 48 h in the 13th, 14th and 15th generations.

Results

The expression level of hTERT protein at the 19th generation in the control group and the 19th and 25th generations in the gene therapy group was significantly different (F = 941.781, P < 0.001). Compared with the 19th generation in the control group, the expression level of hTERT protein at the 19th and 25th generations in the gene therapy group were increased significantly (both P < 0.001). The protein expression levels of IL-6 (t = 61.570, P < 0.001; t = 36.527, P < 0.001) and IL-1α (t = 11.984, P < 0.001; t = 18.622, P < 0.001) at 17th and 19th generations in the gene therapy group were obviously down-regulated as compared with the control group. The expression levels of RAD51 and γ-H2AX proteins of the control group at the 14th generation (t = 3.101, P = 0.036; t = 10.226, P = 0.001) and at the 15th generation (t = 8.683, P = 0.001; t = 7.229, P = 0.002) were much higher than those of the gene therapy group. PDL curve showed that at the 19th generation, the cells in the control group stopped proliferating and were in a completely senescent state, while the cells in the gene therapy group still had proliferative ability (t = 6.856, P = 0.002). After doxorubicin treatment, the expression levels of RAD51 (t = 23.479, P < 0.001; t = 9.619, P = 0.001; t = 14.971, P < 0.001) and γ-H2AX (t = 4.275, P = 0.013; t = 6.787, P = 0.002; t = 9.569, P = 0.001) in the gene therapy group at the 13th, 14th and 15th generations were much lower than those in the control group.

Conclusion

During replicative cellular senescence and doxorubicin-induced cellular senescence, GV3-IL-hT-EGFP fibroblasts can reduce the accumulation of DNA damage and the levels of inflammatory factors by regulating the expression of hTERT after gene therapy.

Key words: Interleukin-6, Cell senescence, Senescent associated secretory phenotype, Telomerase reverse transcriptase, DNA damage

图1 对照组细胞EGFP荧光染色情况（n = 3）注：EGFP.增强型绿色荧光蛋白；IL-6.白细胞介素6；a ~ c图为第10代不同视野下细胞荧光染色情况，d ~ f图为第15代不同视野下细胞荧光染色情况，绿色荧光出现说明IL-6启动子被成功激活；g图为对照组第10代与第15代EGFP阳性细胞比率的比较，与第10代比较，^aP < 0.05

图2 两组细胞hTERT蛋白表达水平的比较（n = 3）注：hTERT.人端粒酶逆转录酶；与对照组第19代比较，^aP < 0.05；与基因治疗组第19代比较，^bP < 0.05

图3 两组细胞第15代、第17代和第19代IL-6和IL-1α蛋白水平的比较（n = 3）注：IL-6.白细胞介素6；C.对照组细胞；T.基因治疗组细胞；与对照组细胞同代比较，^aP < 0.05

图4 两组细胞第13代、第14代和第15代RAD51和γ-H2AX蛋白表达水平的比较（n = 3）注：RAD51.紫外线辐射敏感蛋白；γ-H2AX.磷酸化H2A组蛋白家族成员X；C.对照组细胞；T.基因治疗组细胞；与C组同代细胞比较，^aP < 0.05

图5 两组细胞时间依赖性群体倍增水平的比较（n = 3）注：与对照组细胞第19代比较，^aP < 0.05

图6 经阿霉素诱导衰老的两组细胞第13代、第14代和第15代RAD51和γ-H2AX蛋白表达水平的比较（n = 3）注：RAD51.紫外线辐射敏感蛋白；γ-H2AX.磷酸化H2A组蛋白家族成员X；C.对照组细胞；T.基因治疗组细胞；与C组同代细胞比较，^aP < 0.05

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Interleukin-6 promoter reduces cellular inflammation and DNA damage by regulating human telomerase reverse transcriptase

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Interleukin-6 promoter reduces cellular inflammation and DNA damage by regulating human telomerase reverse transcriptase