摘要
目的构建稳定表达Bcl-2基因的大鼠骨髓间充质干细胞(MSC)载体,观察Bcl-2基因转染对骨髓间充质干细胞生物特性的影响及体外模拟缺血性损伤的保护作用。方法用RT-PCR技术扩增小鼠Bcl-2cDNA,慢病毒介导的基因转导骨髓间充质干细胞,转导后的细胞用Puromycin筛选,RT-PCR法检测转染后Bcl-2的表达,采用血清剥夺和缺氧(SOD)处理建立MSC体外模拟缺血性损伤模型,通过流式细胞术评价转染Bcl-2基因的MSC和未转染的MSC的凋亡及存活情况。对转导Bcl-2的MSC神经诱导及检测。结果转导Bcl-2基因的MSC经Puromycin筛选后可稳定、高效表达Bcl-2蛋白;流式细胞术检测显示:和MSC相比,转导Bcl-2基因的MSC在SOD后凋亡细胞较少,存活细胞较多。转导Bcl-2基因的MSC仍具有向神经细胞分化的潜能。结论慢病毒介导的Bcl-2基因转导MSC有其蛋白的稳定表达,Bcl-2基因转导可对抗MSC体外模拟缺血性损伤引起的细胞凋亡,从而具有保护性作用,且不影响MSC向神经细胞分化的潜能。
Objective To survey the protective effect of B cell lymphoma/leukemia gene 2（Bcl-2）gene-modified mesenchymal stem cells（MSC） injury caused by analogous ischemia in vitro.Methods The fragments of mice Bcl-2 cDNA was amplificated by RT-PCR.Lentivirus-mediated gene transduction was performed and puromycin resistance was adopted for screen,so as to obtain stably transducted cell clones.Bcl-2 protein expression level was evaluated by RT-PCR.Serum and oxygen deprivation was used to cause analogous ischemia injury of MSC in vitro.Flow cytometry were used to evaluate apoptosis and survival of MSC and Bcl-2 gene transducted MSC.The genetically modified MSC underwent nerve cells differentiation and detection.Results MSC transducted with Bcl-2 gene and screend with puromycin showed stable and high effective expression of Bcl-2 protein.The results of flow cytometry showed that the number of apoptosis cell of Bcl-2 transducted MSC was less and the number of survival cell was more than that of non transducted MSC after serum and oxygen deprivation.Bcl-2 transducted MSC retain their differentiation potential into nerve cells.Conclusion Lentivirus-mediated MSC transduction with Bcl-2 gene presented stable Bcl-2 protein expression.Bcl-2 gene transduction can antagonize MSC apoptosis caused by analogous ischemia in vitro thus had protective effect.Bcl-2 transduction doesn＇t affect MSC differentiate into nerve cells.
出处
《中国医科大学学报》
CAS
CSCD
北大核心
2010年第8期599-602,共4页
Journal of China Medical University
基金
国家自然科学基金资助项目(30971018)
关键词
BCL-2基因
基因转导
骨髓间充质干细胞
B cell lymphoma/leukemia gene 2
gene transduction
mesenchymal stem cell
