自噬促进骨髓间充质干细胞复制性衰老过程中衰老相关基因表达  

Autophagy promotes aging-related gene expression during bone marrow mesenchymal stem cell aging

作　　者：吴尚蓉 郑勇 卓儒红[2] 何柳 胡成俊 Wu Shangrong;Zheng Yong;Zhuo Ruhong;He Liu;Hu Chengjun(Department of Anatomy and Embryology, Wuhan University School of Basic Medical Sciences,Wuhan 430071, China;Department of Integrated Medicine,Zhongnan Hospital of Wuhan University, Wuhan 430071, China)

机构地区：[1]武汉大学基础医学院人体解剖学与组织胚胎学系 [2]武汉大学中南医院综合医疗科,武汉430071

出　　处：《中国组织化学与细胞化学杂志》2018年第1期1-7,共7页Chinese Journal of Histochemistry and Cytochemistry

基　　金：国家自然科学基金(81071269;81402296)

摘　　要：目的探究和分析自噬在骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)复制性衰老过程中的作用和可能的调控机制。方法采用全骨髓细胞贴壁法获得大鼠BMSCs,体外连续传代培养至第6代(passage 6,P6),分别用自噬抑制剂巴佛洛霉素A1(bafilomycin A1,Ba F1)和自噬激活剂雷帕霉素(rapamycin,Rap)处理P6 BMSCs。q PCR和Western blot分析细胞自噬活性和衰老相关基因表达的改变,DCFH-DA染色法检测细胞内活性氧(reactive oxygen species,ROS)的含量。结果与增殖活跃的P2 BMSCs相比,P6 BMSCs增殖停滞,Brdu掺入率极低,SA-β-gal染色阳性率高达90%,且P6 BMSCs的p21Waf1和p16ink4a m RNA表达水平明显升高。在P6 BMSCs,ATG12 m RNA表达水平和LC3II/LC3I的比值升高,细胞内ROS含量也增加。对P6 BMSCs采用Ba F1处理阻断自噬流后,p21Waf1和p16ink4a m RNA表达水平降低,ROS含量增加;用Rap处理后,ATG12 m RNA表达水平和LC3II/LC3I蛋白比值均明显升高,且p21Waf1和p16ink4a m RNA表达水平升高,ROS含量降低。结论通过体外连续培养,P6 BMSCs进入衰老状态;自噬可能通过促进衰老相关基因表达调节BMSCs的复制性衰老。Objective To explore the roles and possible mechanisms of autophagy in regulating the aging of bone marrow mesenchymal stem cells(BMSCs).Methods After being isolated from the femora and tibiae of Sprague Dawley rats,BMSCs were cultured in vitro for6passages.They were then treated with autophagy inhibitor baflomycin A(BaF1)or activator Rapamycin(Rap).Cell proliferation and aging sign were measured by Brdu incorporation andβ-galactosidase(SA-β-Gal)staining.qPCR and Western blot were performed to determine the expression of aging and autophagy-associated genes and proteins.The levels of reactive oxygen species(ROS)were measured by DCFH-DA staining.Results Compared to passage2,BMSC proliferation indicated by Brdu incorporation rate significantly reduced at passage6accompanied by increased SA-β-Gal staining and ROS accumulation.At this passage,there was also a significant increase in ATG12,p21Waf1,and p16ink4a mRNA levels as well as LC3II/LC3I ratio compared to BMSCs at passage2.In response to BaF1treatment,it was showed blocking autophagy flux accompanied by decreased p21Waf1and p16ink4a mRNA levels but higher intracellular ROS.In contrast,Rap treatment further increased ATG12,p21Waf1and p16ink4a mRNA levels and LC3II/LC3I ratio but reduced ROS.Conclusion Autophagy may regulate replicative aging of BMSCs through increasing aging related geneexpression.

关 键 词：骨髓间充质干细胞 复制性衰老 自噬 活性氧 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]