携带GFP的慢病毒感染人脐带间充质干细胞及其对Oct4表达的影响.docVIP

携带GFP的慢病毒感染人脐带间充质干细胞 及其对Oct4表达的影响 唐莉, 常静(400016 重庆,重庆医科大学附属第一医院心血管内科) [摘要] 目的 将携带绿色荧光蛋白（GFP）的慢病毒感染人脐带间充质干细胞(hUCMSCs)，并观察其对Oct4表达的影响，为进一步进行携带外源基因的转染探索条件。方法 采用组织块贴壁法分离、培养hUCMSCs; 利用含GFP的慢病毒,以不同 MOI值感染hUCMSCs, 荧光显微镜下观察GFP的表达情况，并通过流式细胞仪（FACS）检测GFP的阳性率，确定最佳MOI值；实验分为未感染组和感染组；采用定量RT-PCR(qRT-PCR)和免疫荧光染色检测细胞中Oct4的表达变化; MTT法检测感染hUCMSCs后对细胞增殖的影响。结果 体外成功分离、培养得到hUCMSCs。荧光显微镜观察在感染96h后荧光表达最强;当以MOI=20感染细胞48h后，GFP阳性率达75%以上。与未感染组相比，qRT-PCR检测Oct4在感染组中的相对表达量为0.9075±0.0124；免疫荧光染色显示Oct4在两组细胞中均有表达且定位于细胞核；MTT结果显示携带GFP的慢病毒对细胞的增殖没有明显影响（P0.05）。 结论 慢病毒携带的GFP基因能够表达于hUCMSCs中，且不影响Oct4的表达,为下一步进行携带外源基因的研究奠定了基础。 [关键词] 绿色荧光蛋白;慢病毒;脐带间充质干细胞;Oct4 [中图法分类号] R373;Q786;R34 [文献标志码] A Infecting human umbilical cord mesenchymal stem cells by lentivirus carrying GFP and its effect on the expression of Oct4 Tang Li, Chang Jing(Department of Cardiology, First Affiliated Hospital , Chongqing Medical University, Chongqing , 400016, China) [Abstract] Objective To infect human umbilical cord mesenchymal stem cells by lentivirus carrying GFP and its effect on Oct4 expression were explored for further exogenous gene transfection. Methods Human umbilical cord mesenchymal stem cells (hUCMSCs) were separated by culture of tissue adherence. HUCMSCs were infected with GFP lentivirus at different virus multiplicity of infection(MOI). GFP expression efficiency was observed using the fluorescence microscope and flow cytometry to obtain optimal MOI value. The experiment was divided into non-transfection group and the GFP transfection group. Oct4 expression in hUCMSCs were measured by Quantitative reverse transcriptase PCR (qRT–PCR) and immunofluorescence staining. Influence of GFP lentivirus on cell proliferation was evaluated by MTT。Results HUCMSCs were successfully isolated in vitro. After infected with GFP lentivirus (MOI=20)for 48 h，GFP positive rate of hUCMSCs was more than 75%. As compared with non-transfection group,the relative expression of Oct4 detected by qRT-PCR in transfection grou