CSE过表达保护低氧/无血清诱导骨髓间充质干细胞凋亡的机制

摘要

目的 探讨胱硫醚-γ-裂解酶(CSE)过表达保护低氧/无血清(H/SD)诱导骨髓间充质干细胞(MSCs)凋亡的机制.方法 体外分离培养大鼠MSCs,利用H/SD诱导MSCs凋亡,构建CSE基因的慢病毒表达载体pLV-ZsGreen-CSE lentivirus及空载体pLV-ZsGreen lentivus并感染MCSs(分别记为CSE MSCs、GFP MSCs).设立正常培养的MSCs(Norm MSCs组)、CSE MSCs组、GFP MSCs组,PI染色流式细胞术检测细胞凋亡率,Western blot法检测CSE、Bcl-2、Bax及细胞色素c(Cyt c)蛋白的表达.结果 与Norm MSCs组及GFP MSCs组相比,CSE MSCs组CSE蛋白表达显著增高(P<0.01),在H/SD 12 h状态下,CSEMSCs组凋亡率显著降低(P<0.01),CSEMSCs组Bcl-2蛋白表达水平显著高于GFP MSCs组及Norm MSCs组(P<0.01),CSEMSCs组Bax蛋白表达水平显著低于GFP MSCs组及NormMSCs组(P<0.01);CSEMSCs组线粒体Cyt c蛋白表达水平显著高于GFPMSCs组及NormMSCs组(P<0.01),CSEMSCs组胞质Cyt c蛋白表达水平显著低于GFP MSCs组及Norm MSCs组(P<0.01).NormMSCs与GFPMSCs组上述指标比较,差异均无统计学意义.结论 CSE过表达保护H/SD诱导MSCs凋亡,其机制与抑制线粒体凋亡途径有关.%Objective To explore the mechanisms of cystathionine-γ-lyase( CSE) overexpression protecting mesen-chymal stem cells(MSCs) from hypoxia and serum deprivation(H/SD)-induced apoptosis. Methods Rat bone marrow MSCs were isolated and cultured in vitro. MSCs were treated with H/SD as an apoptosis model. Plv-Zs-Green-CSE lentivirus and Plv-ZsGreen lentivus vector were constructed and transfected to MSCs( CSE MSCs and GFP M-SCs) . The cells were divided into Norm MSCs group( normal cultured MSCs) ,CSE MSCs group,GFP MSCs group. Apop-tosis of cells was detected by using flow cytometry analysis, Western blot were utilized to determine the protein lev-els of Cyt c,Bcl-2,Bax after H/SD treatment in MSCs. Results CSE overexpression was mediated by lentiviral transduction in MSCs. Compared with GFP MSCs or Norm MSCs group, CSE protein expression in CSE MSCs group was higher markedly(P<0. 01). Afterward, the modified MSCs were treated under H/SD for 12 h. Compared with GF-PMSCs or NormMSCs group. CSEMSCs group had a significant lower proportion of apoptosis(P<0. 01). Bcl-2 protein expressionin CSEMSCs group was markedly upregulated compared with GFPMSCs or NormMSCs group(P<0. 01). Bax protein expression in CSEMSCs group was significantly downregulated compared with GFPMSCs or NormMSCs group(P<0. 01). Furthermore, we found that CSEMSCs group decreased significantly cytochrome c accumulation in the cy-tosol compared with NormMSCs group or GFPMSCs group(P<0. 01). Above parameters were similar between GFPMSCs and Norm MSCs group. Conclusion CSE overexpression protects mesenchymal stem cells from hypoxia and serum deprivation-induced apoptosis via attenuaion of the mitochondrial apoptpsis pathway.