血红素氧合酶-1提高无氧无血清条件下骨髓间充质干细胞的生存  

Transfection of mesenchymal stem cells with human heme oxygenase-1 gene improves survival under conditions of serum-free and hypoxia

作　　者：楚生辉[1] 曾彬[2] 张艳[2] 李杨林 

机构地区：[1]温州医学院药学院,浙江温州325035 [2]武汉大学人民医院心血管内科,湖北武汉430060 [3]湖北省荆州市沙洋县人民医院,湖北沙洋448200

出　　处：《中国病理生理杂志》2009年第6期1206-1209,共4页Chinese Journal of Pathophysiology

基　　金：湖北省卫生厅重点资助项目(No.HB05336)

摘　　要：目的:探讨血红素氧合酶-1(HO-1)是否提高骨髓间充质干细胞(MSCs)在无氧无血清条件下的存活率。方法:取大鼠骨髓,体外分离扩增培养MSCs;HO-1腺病毒转染MSCs,荧光显微镜下观察绿色荧光蛋白(GFP)的表达;DAPI染色观察无氧无血清条件下HO-1-MSCs、MSCs细胞核变化;流式细胞仪检测无氧无血清条件下HO-1-MSCs、MSCs的凋亡率;ELISA检测无氧无血清条件下HO-1-MSCs、MSCs细胞因子的分泌。用无氧无血清条件下HO-1-MSCs、MSCs的培养上清液对无氧无血清条件下的心肌细胞进行培养,Western blot-ting检测凋亡蛋白caspase-3的变化,分光光度计检测caspase-3酶活性。结果:HO-1转染的MSCs可稳定高效地表达GFP;DAPI染色后,MSCs细胞核为典型的凋亡形态,HO-1-MSCs为较均匀蓝色荧光;HO-1-MSCs在无氧无血清条件下的凋亡率显著低于MSCs(P<0.01),且在无氧无血清条件下分泌肝细胞生长因子(HGF)、碱性成纤维细胞生长因子(bFGF)、血管内皮生长因子(VEGF)水平显著增高(P<0.01);HO-1-MSCs、MSCs培养上清液在无氧无血清条件下分别培养心肌细胞,HO-1-MSCs组caspase-3蛋白和caspase-3活性都显著降低(P<0.01)。结论:无氧无血清条件下,HO-1提高MSCs的存活率;HO-1-MSCs分泌更多的细胞因子,且对无氧无血清条件下的心肌细胞有保护作用。AIM ： To investigate the effects of human heme oxygenase - 1 （ HO - 1 ） gene transfection on mesenchymal stem cells （MSCs） survival under the conditions of serum - free and hypoxia. METHODS ： MSCs were acquired from the bone marrow of adult rats. The cells were isolated, purified, cultured, and transfected with Adv - HO - 1. The expression of GFP was detected by immunofluorescence. Cell apoptosis was detected by nuclear DAP! staining and FACS. The concentrations of VEGF, HGF and b - FGF in the culture supernatant were measured by ELISA. The caspase -3 protein level and activity of cardiomyocytes cultured with the supernatants from different MSCs under the condition of serum - free and hypoxia were assayed by Western blotting and fluorimetry, respectively. RESULTS： HO -1 -MSCs exhibited strong expression of GFP. The fragmented or condensed chromatin diminished in HO -1 - MSCs compared with the MSCs lacking exogenous transfeetion of HO - 1 gene. A lower proportion of apoptosis was observed in HO - 1 - MSCs compared with MSCs under the conditions of serum - free and hypoxia （P 〈 0. 01 ）. The expressions of VEGF, HGF and b - FGF in the supernatants of HO - 1 - MSCs were higher than those in MSCs （P 〈 0. 01 ）. A significant reduction of caspase - 3 level and activity in the cardiomyocytes treated with the supernatants from HO - 1 - MSCs was observed, compared to that treated with supernatants from MSCs （P 〈 0. 01 ）. CONCLUSION： HO - 1 improves the MSCs survival under the conditions of serum - free and hypoxia. Several cytokines released by HO -1 - MSCs may protect the cardiomyocytes against ap-optosis.

关 键 词：骨髓间充质干细胞 血红素氧化酶1 细胞凋亡 

分 类 号：R363[医药卫生—病理学]