钠钙交换体启动子促进诱导多能干细胞向心肌细胞转化

背景：诱导多能干细胞治疗缺血性心脏病被认为是极具前景的方法，但目前仍未找到一种理想的诱导方式。目的：探讨钠钙交换体（NCX1）启动子诱导多能干细胞体外分化为心肌细胞的作用。方法：构建含钠钙交换体1启动子的pLVX-IRES-ZsGreen1慢病毒质粒，使用ViraPowerTM慢病毒包装系统共转染293FT细胞。收集病毒测定病毒滴度后感染小鼠诱导多能干细胞，并使用嘌呤霉素进行筛选。观察胚状体数量以及诱导多能干细胞的分化效率，使用RT-PCR、免疫荧光分析法检测心肌特异标记物。结果与结论：成功构建了含钠钙交换体1启动子的pLVX-IRES-ZsGreen1慢病毒，感染小鼠诱导多能干细胞后使用流式细胞仪分选NCX1^＋/GFP^＋细胞。与NCX1^-/GFP-细胞相比，NCX1^＋/GFP^＋细胞在4 d即出现细胞分化和跳动，NCX1＋细胞GATA4/MEF2c/Nkx2.5基因表达分别是NCX1^-细胞的4.2，7.5，2.5倍，免疫荧光显示CX43和心肌肌钙蛋白I呈阳性表达。结果表明，钠钙交换体1启动子可以促进小鼠诱导多能干细胞向心肌细胞分化。

Sodium-calcium exchanger promoter promotes differentiation of mouse induced pluripotent stem cells into cardiomyocytesin vitro

BACKGROUND：Inducing pluripotent stem cels has been considered as a promising treatment for ischemic heart disease. However, an ideal inducing method has not been found yet.
OBJECTIVE：To investigate the role of sodium-calcium exchanger （NCX1） promoter in the differentiation of mouse induced pluriptent stem cels （miPS） into cardiomyocytes.
METHODS： The pLVX-IRES-ZsGreen1 vectors which contain NCX1 promoter constructed by recombinant DNA technology were co-transfected to 293FT cels with ViraPowerTM Lentiviral Packaging Mix. The recombinant lentiviruses infected with miPS were selected and purified by puromycin. miPS were recovered and passaged to form embryoid bodies. The embryoid bodies were induced by differentiation medium containing various concentrations of the virus titer. The number of beating embryoid bodies were calculated. The expression profiles of the myocardial intra-markers were tested to determine the differentiation efficiency of iPSC by RT-PCR and immunofluorescence analysis.
RESULTS AND CONCLUSION：pLVX-IRES-ZsGreen1 vectors which contain NCX1 promoter were constructed and confirmed by PCR. Virus could be packaged, purified and concentrated successfuly. The recombinant lentivirus to transduce miPS was sorted by flow cytometry. In contrast to NCX1^-/GFP^- cels, NCX1^＋/GFP^＋ cels were differentiated and developed prominent beating areas with sustained contractile activity for additional 4 days, and demonstrated positive expression of gap communication marker CX43 and cardiac troponin. The expressions of GATA4, MEF2c and Nkx2.5 in the NCX1^＋ cels were 4.2, 7.5, and 2.5 times those in NCX1^- cels. Results showed the NCX1 promoter can promote the cardiac differentiation of miPS .