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  

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

作　　者：刘洋 盛赵莹 孙琳琳 Liu Yang;Sheng Zhaoying;Sun Linlin(Department of Geriatrics,the First Affiliated Hospital,Zhejiang University School of Medicine,Key Laboratory of Diagnosis and Treatment of Aging and Physic-chemical Injury Diseases of Zhejiang Province,Hangzhou 310003,China)

机构地区：[1]浙江大学医学院附属第一医院老年病科、浙江省增龄与理化损伤性疾病诊治研究重点实验室,杭州310003

出　　处：《中华危重症医学杂志（电子版）》2024年第2期104-110,共7页Chinese Journal of Critical Care Medicine:Electronic Edition

基　　金：国家自然科学基金青年科学基金项目(82001464)。

摘　　要：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=9.56Objective: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-1αproteins were detected at the 15th,17th and 19th generations.At the same time,the protein expression levels of RAD51 andγ-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

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 

分 类 号：R339.38[医药卫生—人体生理学]