雷帕霉素对衰老细胞生物节律关键基因的调控作用观察

摘要

Objective To investigated the effect of rapamycin on the cell autonomous circadian rhythm of senescent cells. Methods We induced replicative senescence of human dermal fibroblasts by series passage. The senescent human dermal fibroblasts cells were identified by SA-β-gal staining. The senescent human dermal fibroblasts cells were synchro-nized by 50% horse serum,and treated with DMSO or 200 nmol/L rapamycin. The oscillation profiles of BMAL1,PER2, CRY1,Rev-erbα and Rorα were examined by fluorogenic quantitative PCR. The circadian rhythm of genes was analyzed by JTK-CYCLE software. The protein expression of BMA1,CRY1,and PER2 was detected by Western blotting. Results The mRNA expression of PER2,CRY1,Rev-erbα and Rorα in the observation group was higher than that of the control group (all P <0.05). The biorhythm cycle of BMAL1,CRY1,Rev-erbα,and Rorα genes in the control group was 24 h, and the biorhythm cycle of PER2 gene was 28 h. The biorhythm cycle of BMAL1,CRY1,and Rorα genes in the observa-tion group was 24 h,and the biorhythm cycle of Rev-erbα gene was 27.5h. The expression of CRY1 protein in the obser-vation group was significantly higher than that in the control group,while PER2 protein expression was significantly lower than that in the control group (both P < 0.05). No significant difference was found in BMAL1 mRNA and protein expres-sion between these two groups. Conclusion Rapamycin can increase the expression of BMAL1,PER2,CRY1,Rev-erbαand Rorα mRNA in the aging human dermal fibroblasts and shorten the rhythmic cycle of PER2,which plays a regulatory role in the disturbance of circadian clock function.%目的 观察雷帕霉素对衰老细胞生物节律关键基因的调控作用.方法 通过传代诱导人皮肤成纤维细胞复制性衰老,获得衰老的人皮肤成纤维细胞.加入50%的马血清刺激诱导细胞同步化,将细胞分为观察组和对照组,分别加入100 mmol/L的雷帕霉素和DMSO培养24 h.采用荧光定量PCR法检测生物节律关键基因芳烃受体核转位蛋白3(BMAL1)、节律周期蛋白2(PER2)、蓝光受体蛋白1(CRY1)、孤儿核受体α(Rev-erbα)、孤核受体α(Rorα)mRNA表达,利用JTK-CYCLE软件分析各基因的节律周期,Western blotting法检测BMAL1、PER2、CRY1蛋白表达.结果 观察组PER2、CRY1、Rev-erbα、RorαmRNA表达高于对照组(P均<0.05).对照组BMAL1、CRY1、Rev-erbα、Rorα基因的节律周期为24 h,PER2基因的节律周期为28 h.观察组BMAL1、CRY1、Rorα基因的节律周期为24 h,Rev-erbα基因的节律周期为27.5 h,PER2的节律周期为24 h.观察组CRY1蛋白表达高于对照组(P<0.05),PER2蛋白表达低于对照组(P<0.05),两组BMAL1 mRNA及蛋白表达比较无统计学差异.结论雷帕霉素可以提高衰老的人皮肤成纤维细胞生物节律关键基因BMAL1、PER2、CRY1、Rev-erbα、RorαmRNA的表达,缩短PER2的节律周期,对紊乱的节律钟功能具有调控作用.