MnSOD过表达对t-BHP诱导间充质干细胞凋亡的保护作用  

Protective Effects of Overexpression of MnSOD on t-BHP Induced Apoptosis in Mesenchymal Stem Cells

作　　者：杨超[1] 戴为民[2] 张晓梅[3] 陈海旭[4] 杨博[2] 宫媛[1] 王昌正[1] 车宇芳[1] 吴本俨[1] 

机构地区：[1]解放军总医院南楼消化内科,北京100853 [2]解放军总医院胸外科,北京100853 [3]解放军总医院消化内科,北京100853 [4]解放军总医院老年医学研究所,北京100853

出　　处：《生物化学与生物物理进展》2013年第6期556-564,共9页Progress In Biochemistry and Biophysics

基　　金：国家自然科学基金(B1070296);中国博士后科学基金(2011M500155)资助项目~~

摘　　要：通过重组慢病毒系统感染人间充质干细胞(mesenchymal stromal cells,MSCs),建立了能够稳定、高效表达锰超氧化物歧化酶(MnSOD)的细胞株MnSOD-MSCs.从胎儿肝脏组织克隆MnSOD基因,构建重组慢病毒MnSOD的表达载体,感染MSCs.根据荧光表达进行流式分选,获得能够继续稳定传代的高表达MnSOD基因的MSCs,RT-PCR和Western blot结果证实细胞株中的MnSOD基因稳定高表达.用不同浓度的第三丁基过氧化氢(t-BHP)处理细胞,通过CCK-8法检测细胞的存活率,SA-β-gal染色观察细胞的衰老情况,流式细胞技术分析细胞的凋亡率,实时荧光定量PCR分析p53和p53正向细胞凋亡调控因子(PUMA)的表达.结果发现,MnSOD过表达可提高细胞的存活率,抑制细胞的凋亡,SA-β-gal染色阳性率降低,且p53和PUMA表达下调.这提示MnSOD过表达对t-BHP诱导的细胞凋亡具有保护作用.The study was aimed to investigate the effects and mechanism of manganese superoxide dismutase （MnSOD） on tert-butyl hydroperoxide（t-BHP） induced apoptosis in human bone marrow mesenchymal stem cells （MSCs）. The MnSOD gene was cloned from human fetal liver by RT-PCR. The MnSOD recombinant plasmid was transfected into MSCs stably by lentiviral system. The efficiency of virus transfection was identified by expression of enhanced green fluorescence protein （EGFP） analyzed by fluorescence microscope, then MSCs were sorted by fluorescence-activated cell sorting （FACS） according to strong EGFP expression. MnSOD expression was detected by RT-PCR and Western blot. Transfected cells were then treated with different concentration of t-BHP, and the features including cell viability, senescence-associated [3-gal activity, and apoptosis were evaluated. Our data demonstrated that overexpression of MnSOD could promote MSCs viability by inhibiting apoptosis or cellular senescence. Furthermore, apoptosis related genes p53 and PUMA were down-regulated. Therefore, these results indicated that overexpression of MnSOD in MSCs could protect against t-BHP induced apoptosis.

关 键 词：MNSOD 基因转染 间充质干细胞 t-BHP 凋亡 

分 类 号：Q554[生物学—生物化学] Q786