大鼠骨髓间充质干细胞转染GATA-4的表达及鉴定  

Expression and identification of rat bone marrow mesenchymal stem cells overexpressing GATA-4 gene

作　　者：李红霞[1] 周亚峰[1] 蒋彬[1] 陈弹[1] 杨向军[1] 蒋文平[1] 

机构地区：[1]苏州大学附属第一医院心内科,苏州医学博士研究生215006

出　　处：《医学研究生学报》2013年第3期228-231,共4页Journal of Medical Postgraduates

基　　金：国家自然科学基金(81100173);江苏省普通高校研究生科研创新计划(CXZZ11_0118)

摘　　要：目的基因修饰间充质干细胞(mesenchymal stem cell,MSC)是增加细胞抗缺氧的新方向。文中通过建立稳定的基因重构MSC细胞模型研究GATA-4转染骨髓MSC的特征及鉴定,为GATA-4修饰的MSC应用于急性心肌梗死等心肌疾病的治疗提供有效的技术手段。方法分离、培养及纯化新生大鼠MSC,用流式细胞仪进行鉴定。将培养第3代的MSC用pMSCV逆转录病毒表达系统转染GATA-4基因(MSCGATA-4),对照组为空质粒组(MSCNull)。用RT-PCR、Western blot印迹和免疫组化鉴定转染的效果。结果大鼠MSC表面抗原CD45和CD44表达阴性,CD31表达弱(<3%),CD90表达强阳性,其阳性率达95%。MSCGATA-4组GATA-4 mRNA和蛋白表达明显高于对照组MSCNull。MSCGATA-4组GATA-4和GFP都显示阳性,而对照组只表达GFP。结论 GATA-4成功转染到MSC,这将为研究GATA-4对MSC抗缺氧作用奠定基础。Objective Gene modified MSCs is a new investigation direction to increase cell resistance to hypoxia. The objec- tive of this study was to investigate GATA-4 gene expression and identification of mesenchymal stem cells （MSCs） by means of the cont- rusting stable GATA-4 gene recontruction cell model to provide effective measurements for the treatment of myocardial diseases, such as acute myocardiac infarct. Methods MSCs were isolated and cultured from the bone marrow of new-boru rat. Rat MSCs were identi- fied by flow cytometry. MSC were transfected with GATA-4 （MSCCATA4） using a murine stem cell virus （pMSCV） retroviral expression system at the third passage. Control cells were transduced with empty vector （MSCNull）. Gene expression in MscGATA-4was analyzed using real-time quantitative polymerase chain reaction （real-time PCR）, Western blotting and Results Rat MSCs were negative for markers for hematopoietic cells （ CD45 ） and markers for T cells, B ceils, macrophages and thymocytes （CD44）. Rat MSCs were dimly positive for CD31 （ 〈 3% ）, a marker for endothelial cells, platelets and leucocytes. However, MSCs were strongly positive for CD90 （ 〉 95% ）, a marker for stem cell. Expression of GATA-4 mRNA and protein were higher in the group of MSCGAT-4 than control group MSCNull. GATA-4 and GFP were positive in the group of MSCGATA＇4, while only GFP was expressed in control group. Conclusion GATA-4 was succefully transfected into MSC that will paly a foundation to further investigate its role of anti-hypoxia.

关 键 词：骨髓间充质干细胞 GATA4 基因转移技术 基因治疗 

分 类 号：Q26[生物学—细胞生物学]