摘要
目的探讨microRNA(miRNA)和衰老心肌细胞自噬之间的关系。方法使用0、4、8、16、20 g/L D-半乳糖刺激大鼠心肌细胞H9c2衰老,刺激3、6、9天后,β-半乳糖苷酶染色法检测细胞衰老水平;电镜观察D-半乳糖刺激衰老大鼠心肌细胞自噬体形成;分别采用实时荧光定量PCR(qRT-PCR)及蛋白质印迹法(Western blot)检测衰老大鼠心肌细胞与正常H9c2细胞中miRNA-30a、miRNA-126、miRNA-204及自噬蛋白LC3B II/LC3B I表达水平。结果与对照组比较,随着药物浓度的升高及刺激天数的增加,细胞染色逐渐加深,在D-半乳糖浓度为8 g/L刺激9天时,细胞染色最明显;电镜结果显示,D-半乳糖诱导衰老心肌细胞自噬体减少;Western blot结果显示,衰老心肌细胞LC3B II/LC3B I水平降低;实时荧光定量PCR结果显示,诱导衰老组心肌细胞中,miRNA-30a、miRNA-126表达水平明显下降,miRNA-204表达水平明显上升。结论 D-半乳糖诱导的衰老心肌细胞自噬水平降低的过程中,存在miRNA-30a、miRNA-126表达水平的降低以及miRNA-204表达水平的增加,miRNA的差异表达可能是衰老心肌细胞自噬水平变化的重要机制。
Aim To investigate the relationship between microRNA (miRNA) and autophagy in aged myocardial cells induced by D-galactose. Methods The D-galactose with 0, 4, 8, 16, 20 g/L was used to induce the rat myocardial cells to senescence respectively,and being stimulated 3, 6, 9 days later, the aging level of H9c2 was detected by the beta-galactosidase staining; the formation of autophagosomes in aging rat cardiomyocytes induced by D-galactose was detected by electron microscope; the mRNA expression level of miRNA-30a, miRNA-126, miRNA-204 and protein expression level of LC3B Ⅱ/LC3B Ⅰ in aging rat cardiomyocytes and H9C2 cells was detected by quantitative real-time(qRT-PCR) and Western blot methods respectively. Results Compared with the control group, the cell staining was gradually deepened with the increase of drug concentra tion and the stimulation days, and when D-galactose concentration was 8 g/L for 9 days, cell staining was the most obvious ; electron microscopy showed that the autophagy in aging cardiomyocytes induced by D-galactose was decreased; Western blot results showed that the level of LC3B Ⅱ/LC3BⅠ in aging eardiomyocytes was reduced; qRT-PCR results showed that in the aging cardio- myocytes group the expression levels of miRNA-30a and miRNA-126 were decreased and miRNA-204 was significantly increased. Conclusion In the process of the autophagy level decreasing in senescent cardiomyocytes induced by D-galactose, there exists a reducing in miRNA-30a, miRNA-126 expression level and an increasing in miRNA-204 expression level, the differential expression of miRNAs may be an important mechanism of autophagy in senescent cardiomyocytes.
作者
朱光荣
丁宁
杨安宁
杨松昊
王楠
侯苗苗
谢琳
蒋袁絮
田珏
姜怡邓
杨晓玲
ZHU Guang-Rong DING Ning YANG An-Ning YANG Song-Hao WANG Nan HOU Miao-Miao XIE Lin JIANG Yuan-Xu TIAN Jue JIANG Yi-Deng YANG Xiao-Ling(School of Basic Medicine, Ningxia Medical University, Yinchuan, Ningxia 750004, Chin)
出处
《中国动脉硬化杂志》
CAS
北大核心
2017年第4期332-336,共5页
Chinese Journal of Arteriosclerosis
基金
国家自然科学基金资助项目(81560242
81360063)
宁夏教育厅项目(NGY2015092)