【摘要】 目的探讨基因修饰的方法纯化人脐带间充质干细胞源性心肌细胞的可行性,为临床心肌细胞移植寻找新的干细胞来源。方法将人心肌球蛋白轻链(MLC-2v)基因启动子与嘌呤霉素抗性基因融合,然后转染人脐带间充质干细胞,通过嘌呤霉素筛选,纯化培养出MLC-2v阳性细胞,用5-氮杂胞苷进行诱导,检测诱导后MLC-2v阳性细胞是否表达心脏相关标记物。结果纯化培养出的MLC-2v阳性细胞经5-氮杂胞苷诱导后表达心肌细胞的标记物肌钙蛋白Ⅰ(troponinⅠ)、MLC-2v和结蛋白(desmin)。RT-PCR检测证实,人脐带间充质干细胞在转染及诱导前不表达Nkx2.5和desmin,经筛选诱导后细胞表达Nkx2.5和desmin。结论人脐带间充质干细胞通过基因修饰方法可以得到相对纯化的心肌样细胞,可能成为心脏细胞移植治疗重要的细胞来源。更多还原

【Abstract】 Objective To investigate the feasibility of purification of human umbilical cord mesenchymal stem cell-derived cardiomyocytes by genetic modification,as a new source of stem cells for clinical myocardial cell transplantation. Methods First of all,the human cardiac myosin light chain ( MLC-2v ) gene promoter and puromycin resistance gene were fused by genetic modification,and then the human umbilical cord mesenchymal stem cells were transfected. Secondly,the MLC-2V-positive cells were purified by puromycin and then the cells were cultured. After that,the MLC-2v-positive cells were induced by 5-azacytidine and the expression of cardiac markers were detected. Results The expression of myocardial cell markers troponin I ( troponin I) , MLC-2v and desmin could be detected in the green fluorescent protein-expressing MLC-2v cells induced by 5-azacytidine. RT-PCR analysis showed that human umbilical cord mesenchymal stem cells expressed the mRNA of Nkx2. 5 and desmin after transfection and induction,which were absence without transfection and induction. Conclusions Human umbilical cord mesenchymal stem cells can be induced to relatively purified cardiomyocyte-like cells by genetic modification,which will become the important source of cardiomyocytes for transplantation in patients.更多还原