调控Notch信号通路对胚胎干细胞向心肌细胞分化的影响  

Effects of Notch Signaling Pathway on the Differentiation of Embryonic Stem Cells to Cardiomyocytes

作　　者：胡家芸[1] 徐红新[1] 田毅浩[2] 曲折[1] 黄文蔚[1] 蒋学俊[1] 李庚山[1] 

机构地区：[1]武汉大学人民医院心内科,湖北武汉430060 [2]武汉大学基础医学院解剖学教研室,湖北武汉430071

出　　处：《武汉大学学报（医学版）》2013年第4期485-489,共5页Medical Journal of Wuhan University

基　　金：国家自然科学基金资助项目(编号:30700314);武汉市晨光计划资助项目(编号:20065004116-02)

摘　　要：目的:观察Notch信号通路调控对胚胎干细胞向心肌细胞分化的影响。方法:实验分为两部分:调控Notch信号通路开关,研究其对胚胎干细胞向心肌细胞分化比例的影响及心肌分化相关蛋白表达的影响。在小鼠胚胎干细胞培养的基础上,加入Notch信号激活剂Jagged蛋白(5mg/L)或Notch信号抑制剂MW167(100μmol/L)作用14d,检测:①自发性节律收缩细胞的出现情况;②心肌特异性收缩蛋白的表达情况,计算胚胎干细胞分化为心肌细胞的比例;③采用Westernblot方法检测胚胎干细胞源性心肌细胞中心肌分化相关蛋白(Nkx2.5、GA-TA4、Tbx5、Myocardin)的表达。结果:与对照组(胚胎干细胞自发分化组)相比,加入Jagged蛋白后,胚胎干细胞向心肌细胞分化的比例和早期心肌分化相关蛋白表达水平均显著减少;而加入MW167后,分化比例及早期心肌分化相关蛋白均显著增加。结论:胚胎干细胞中加入Jagged蛋白持续激活Notch信号,抑制了胚胎干细胞向心肌细胞分化;而加入MW167持续抑制Notch信号,则促进了胚胎干细胞向心肌细胞分化。Objective： To observe the effects of Notch signaling pathway on the differentiation of embry- onic stem cells to myocardial cells. Methods： The experiments were divided into two parts： regu- lating the switch of Notch signaling pathway to study the effects on differentiation rates of em bryonic stem cells to cardiomyocytes, and determining the expression of myocardium differentia- tion related proteins. Mouse embryonic stem cells were labeled with DAPI fluorescence （200 μg/ ml）. Then Jagged protein （5 rag/L）, an activator of notch signal, or MW167 （100 μmol/L）, an inhibitor of notch signal, was added to culture embryonic stem cells for 14 days. The appearance of spontaneously contractive ceils was observed under phase contrast microscope. The expression of myocardial specific contractive protein was identified by cell immunofluorescence assay, and the differentiation ratio of embryonic stem cells to cardiomyocytes was calculated. The expression of myocardial differentiation related proteins were identified by Western blot. Results. At the 14th day, there were about 15% embryonic stem cells differentiated into spontaneously contrac- tive cardiomyocytes, and the differentiation ratio was significantly reduced with Jagged treat- ment, whereas dramatically increased with MW167 treatment. Western blot results suggested that the expression of early myocardial differentiation related proteins were significantly reduced when cells were treated with Jagged, whereas dramatically increased when the ceils were treated with MW 167. Conclusion. Jagged protein inhibits the differentiation of embryonic stem cells to cardiomyocytes through activating notch signaling pathway, however, MW167 can promote the differentiation of embryonic stem cells to cardiomyocytes through inhibiting notch signaling path- way.

关 键 词：心肌细胞 胚胎干细胞 Jagged蛋白 MW167 NOTCH 

分 类 号：Q813.11[生物学—生物工程]