小鼠胚胎房室管内皮细胞EMT体外研究方法的改良

Experimental method improvement for endocardial-mesenchymal transformation of mouse embryonic atrioventricle canal endothelial cells in vitro

目的:对小鼠胚胎房室通道内皮细胞向间充质细胞转化(epithelial-mesenchymal transdifferentiation,EMT)体外研究方法的改良。方法:根据小鼠胚胎房室管内皮细胞EMT体外胶原凝胶实验方法进行改良,去胚胎时以37℃的1×M199培养基代替冰PBS,培养液中加入了ITS。结果:显微镜下观察培养8h内皮细胞贴着胶原表面生长;48h后,爬出的间充质细胞数量和爬行的距离达到顶峰;细胞PECAM-1阳性信号的细胞与SMA阳性信号的细胞没有共定位;爬入胶原的细胞数量比率平均达(95.7±1.4)%。结论:本方法介绍的小鼠胚胎房室通道EMT体外研究方法,是很理想的体外实验的模型,可以满足各种心脏瓣膜发育实验研究的要求,为先天性心脏病的研究提供有力的实验平台。

Objective To improve the experimental method for endocardial-mesenchymal transformation （EMT） of mouse embryonic atrio-ventricle canal（AVC） endothelial cells in vitro. Methods The method of using ex vivo explant culture system for EMT of AVC endothelial cells was improved as follows ： first, the process of the embryonic dissection was incubated in the 1 ×M199 medium at 37℃. Second, ITS was added into the cell culture medium. Results In the AVC endothelial cells ex vivo explant culture system, the mesenchymal cells were found proliferated and migrated into the cardiac gel at 8 hours after explanting. About 48 hours later, the amount of mesenchymal cells reached the top of the EMT. The ratio of the mesenchymal cells in the gel was close to （95.7 ±1.4） %. Conclusion The improved method of ex vivo explant culture system for EMT of AVC endothelial ceils is very useful for heart valve research.