原代大鼠主动脉内皮细胞衰老模型的建立  

Establishment of senescent cell model in primary rat aortic endothelial cells

作　　者：苗新宇[1] 朱潇潇 龚燕平[1] 谷昭艳[1] 李春霖[1] MIAO Xinyu;ZHU Xiaoxiao;GONG Yanping;GU Zhaoyan;LI Chunlin(Department of Endocrinology, Second Medical Center, PLA General Hospital, Beijing 100853, China)

机构地区：[1]解放军总医院第二医学中心内分泌科,北京100853

出　　处：《细胞与分子免疫学杂志》2019年第3期230-235,共6页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(81774119)

摘　　要：目的建立血管衰老模型,观察比较高葡萄糖(HG)、血管紧张素Ⅱ(AngⅡ)、过氧化氢(H_2O_2)及棕榈酸(PA)对原代大鼠主动脉内皮细胞(RAEC)诱导衰老作用。方法取2月龄雄性Wistar大鼠,组织贴块法提取原代RAEC并采用免疫荧光细胞化学染色检测CD31的表达并进行鉴定。分别应用30 mmol/L葡萄糖(HG)、 10μmol/L AngⅡ、 100μmol/L H_2O_2、 0.5 mmol/L PA处理原代RAEC 24 h。采用β-半乳糖苷酶染色观察细胞衰老情况,实时荧光定量PCR检测衰老相关基因P16 mRNA的水平, Western blot法检测衰老相关基因P16、 P21、 P53蛋白水平;免疫荧光细胞化学染色观察P16的表达, CCK-8法观察细胞存活率。结果与对照组相比,各处理组RAEC中β-半乳糖苷酶染色细胞增加,以H_2O_2、 PA处理组更明显; HG、 AngⅡ、 H_2O_2、 PA处理均可增加RAEC的P16 mRNA水平; AngⅡ、 H_2O_2、 PA处理均可增强RAEC的P16、 P21蛋白水平,以H_2O_2组最显著。各处理组细胞P16表达均增强,与对照组相比, HG组与AngⅡ组细胞存活率变化不大, H_2O_2组与PA组细胞存活率显著降低。结论 HG、 AngⅡ、 H_2O_2及PA均可诱导建立RAEC衰老模型,以H_2O_2诱导衰老作用最强。Objective To establish senescent models in rat aortic endothelial cells(RAECs) induced by high glucose(HG), angiotensin Ⅱ(AngⅡ), hydrogen peroxide and palmitic acid(PA), and compare the senescence-induced effects of these factors. Methods Primary RAECs were extracted from two-month-old male Wistar rats by issue explant method and identified by CD31 immunofluorescence cytochemistry. RAECs were treated separately by 30 mmol/L(HG), 10 μmol/L AngⅡ, 100 umol/L hydrogen peroxide(H2O2) and 0.5 mmol/L PA. Twenty-four hours later, senescence-associated β-galactosidase(SA-β-gal) staining was used to evaluate the senescent state. Real-time quantitative PCR was used to investigate mRNA expression level of senescence-related gene P16. Western blot analysis was performed to determine protein expression levels of P16, P21 and P53. Immunofluorescence cytochemistry was used to detect the expression of P16 protein in cells. The cell viability of RAECs was tested via CCK-8 assay. Results Compared with the control group, positive rate of SA-β-gal staining in each treatment group increased, especially in H2O2 and PA groups. And mRNA expression level of P16 increased in all four groups. P16 and P21 proteins had high expression in AngⅡ, H2O2 and PA groups, most obviously in H2O2 group. P16 immunofluorescence expression level was enhanced in all groups. The cell viability in HG and AngⅡ groups was similar with the control group, while H2O2 and PA groups had low cell viability. Conclusion The aging mode of RAECs is successfully established by HG, AngⅡ, H2O2 or PA treatment, and H2O2 treatment shows the strongest effect.

关 键 词：衰老 大鼠主动脉内皮细胞(RAEC) 原代培养 

分 类 号：R392-33[医药卫生—免疫学] R543.1[医药卫生—基础医学]