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.
出处
《基础医学与临床》
CSCD
北大核心
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)
