摘要
目的探讨Nrf2调节氧化应激状态下对骨髓间充质干细胞(Bone marrowmesenchymal stem cells,BMSCs)凋亡的作用及机制。方法原代培养SD大鼠BMSCs;构建过表达Nrf2重组慢病毒载体(pLV-hefla-EGFP)和沉默Nrf2重组慢病毒载体(pLV-Hu6-Nfe212 shRNA02-hEFLa-EGFP),并以空载体质粒pLV-hefla-EGFP、基因沉默质粒pLV-Hu6-shRNA02-hEFla-EGFP作为实验空载体阴性对照,分别转染骨髓间充质干细胞,建立稳定过表达Nrf2及沉默Nrf2的骨髓间充质干细胞系;H 2O 2模拟心肌梗死后氧化应激状态;随机分为5组:空白对照组(Control)、Nrf2干扰空载体组(Nrf2-Vector)、Nrf2干扰组(si-Nrf2)、Nrf2过表达空载体组(Nrf2-Scramble)、Nrf2过表达组(LV-Nrf2)。Western Blot鉴定慢病毒转染Nrf2 BMSCs成功;Annexin V-FITC/PI双标记流式细胞术检测BMSCs凋亡率;Western Blot检测Nrf2、HO-1、NQO1等抗氧化基因蛋白的表达。结果Control组、si-Nrf2组、LV-Nrf2组总体凋亡率(%)分别为68.52±0.04、89.22±0.02、38.99±0.06;与Control组相比,LV-Nrf2组BMSCs的总体凋亡率显著降低(P<0.05),si-Nrf2组BMSCs总体凋亡率显著升高(P<0.05);此外,与Control组相比,LV-Nrf2组抗氧化相关蛋白Nrf2、HO-1、NQO1相对表达量显著上调(P<0.05);si-Nrf2组抗氧化相关蛋白Nrf2、HO-1、NQO1相对表达量显著下调(P<0.05)。结论过表达Nrf2可有效减少氧化应激状态下骨髓间充质干细胞的凋亡,其机制可能是通过激活Nrf2/HO-1/NQO1信号通路。
Objective To investigate the mechanisms underlying Nrf2 regulated apoptosis of Bone Marrow Mesenchymal Stem Cells(BMSCs)under oxidative stress.Methods Primary BMSCs culture was from SD rat.Lentivirus transfected BMSCs was established.The oxidative stress state after myocardial infarction was stimulated by H 2O 2.This study was randomly divided into five groups:Control group;the overexpression of Nrf2 group(LV-Nrf2);the empty vector overexpressed of Nrf2 group(Nrf2-Scramble);the interference of Nrf2 group(si-Nrf2);the interfered with empty Vector of Nrf2 group(Nrf2-Vector).Western blot assay identified the transfection of Nrf2 BMSCs with lentivirus.Annexin V-FITC/PI double-labeled flow cytometry was used to detect the apoptosis rate of BMSCs.Western blot assay was used to detect the expression of Nrf2,HO-1,NQO1 and other related anti-oxidant proteins.Results Compared with Control group,the overall apoptosis rate of BMSCs in LV-Nrf2 group was significantly reduced.The overall apoptosis rate of BMSCs in si-Nrf2 group was increased.In addition,compared with Control group,the expression levels of anti-oxidant genes and related proteins Nrf2,HO-1 and NQO1 in LV-Nrf2 group were increased.The expression levels of anti-oxidant genes and related proteins Nrf2,HO-1 and NQO1 in si-Nrf2 group were decreased.Conclusion Overexpression of Nrf2 could effectively reduce the apoptosis of BMSCs under oxidative stress,and its mechanism may be through activating of Nrf2/HO-1/NQO1 signaling pathway.
作者
汤娅
石蓓
赵然尊
Tang Ya;Shi Bei;Zhao Ranzun(Department of Cardiology,Affiliated Hospital of Zunyi Medical University,Zunyi Guizhou 563099,China)
出处
《合肥医科大学学报》
2019年第5期509-516,共8页
Journal of Zunyi Medical University
基金
国家自然科学基金资助项目(NO:81660049)
贵州省科技厅社发处项目(NO:黔科合SY字[2013]3046)。
