小鼠Islet-1基因慢病毒表达载体的构建及其诱导C_3H_(10)T1/2细胞向心肌样细胞特异性分化  

Construction of lentiviral expression vector expressing mouse Islet-1 gene and investigation of Islet-1 mediated induction of C_3H_(10)T1/2 cell differentiation into cardiomyocyte-like cells

作　　者：智深深[1] 田杰[2] 刘官信[1] 鲁荣[1] 林建萍[1] 刘建平[1] 朱静[1] 

机构地区：[1]重庆医科大学儿童医院儿童发育疾病研究省部共建教育部重点实验室,重庆400014 [2]重庆医科大学儿童医院心血管内科,重庆400014

出　　处：《基础医学与临床》2011年第7期740-745,共6页Basic and Clinical Medicine

基　　金：国家自然科学基金(30973219);自然科学基金重点项目(CSTC;2009BA5084)

摘　　要：目的研究Islet-1对干细胞分化的影响。方法用PCR钓取目的基因,将目的基因与pLenO-WPI载体连接,选取阳性质粒,与辅助质粒共同感染293T细胞生产出慢病毒载体。感染C3H10T1/2细胞,实时荧光定量PCR及Western blot检测Islet-1和心肌、肝脏、骨骼及神经各系统相关标志物的表达,免疫荧光检测心肌肌钙蛋白T(cTnT)表达部位。结果 PCR及测序显示目的片段正确插入,实验组有Islet-1表达;心肌早期发育相关基因GATA-4、MEF2C、NKx2.5在检测到荧光蛋白1周后升高,2周到达高峰,3周后可检测到心肌特异性蛋白cTnT(0.582±0.0576),其时序性表达呈随时间增强趋势;cTnT表达于胞质;肝脏系统特异性标志AFP及ALB、骨骼系统特异性标志BGP及BALP、神经系统特异性标志Nestin及GFAP均未表达。结论 Islet-1具有特异性促进干细胞向心肌样细胞分化的作用。Objective To construct lentiviral expression vectors targeting mouse gene Islet-1 and to study the effect of Islet-1 on stem cell differentiation.Methods Islet-1 gene was obtained by PCR and inserted into pLenO-WPI Vector.The positive plasmid was selected and infected 293T cells with helper plasmid to produce recombinact lentivirus.Islet-1 expression and its related gene,gene and protein markers of heart,liver,bone,and nerves systems were measured by Real-time quantitative PCR and Western blot after C3H10T1/2 was infected by the lentiviruses.Results PCR and sequencing showed that the right DNA fragment and Islet-1 had been inserted and detected in both in gene and protein levels.Cardiac development related genes GATA-4,MEF2C;NKx2.5 increased in 1 week and reached the highest level in 2 weeks,and cTnT reached a high level in 3 weeks and increasedover time.There were no expressions of gene and protein markers of liver,bone,brain.Conclusion Lentiviral expression vectorcarrying mouse Islet-1 gene was constructed.Islet-1 promoted C3H10T1/2 cells differentiate into cardiomyocyte-like cells specifically,that laid the foundation for further investigation of Islet-1.

关 键 词：慢病毒表达载体 Islet-1 干细胞特异性分化 心肌样细胞 

分 类 号：R346[医药卫生—基础医学]