TNNI3K基因过表达诱导小鼠胚胎干细胞向心肌细胞分化

The Overexpression of Cardiomyocyte-specific Kinase TNNI3K Induces Mouse Embryonic Stem Cells Differentiation into Cardiomyocytes

目的研究肌钙蛋白Ⅰ相关激酶(TNNI3K)对小鼠胚胎干细胞(m ESC)向心肌细胞分化的影响。方法从形态和细胞免疫荧光、碱性磷酸酶试验以及HE染色鉴定m ESC的多能性。采用悬滴法培养m ESC形成拟胚体(EB),自分化为跳动的心肌细胞,通过细胞免疫荧光和透射电镜(TEM)方法鉴定。慢病毒分别携带h TNNI3K基因和siRNA感染m ESC,并分别自分化为心肌细胞,通过流式细胞术(FCM)、细胞免疫荧光、Western blot等分析心肌特异性蛋白表达水平的差异。结果 m ESC表面蛋白分子SSEA-1和Oct-4表达呈阳性(绿色),ALP试验呈蓝紫色,核质比>>1。正常自分化的细胞免疫荧光显示cTnⅠ、cTnT、MLC2以及α-actinin阳性(绿色),肌节清晰可见。TEM显示心肌细胞独有的肌纤维亚细胞结构。过表达组cTnT+阳性细胞率高于对照组,Western blot显示心肌特异性蛋白cTnT、cTnⅠ、MLC2、α-actinin等显著高于对照组,且细胞免疫荧光结果显示MHC6蛋白提前表达。干扰组不仅cTnT+阳性细胞率显著低于对照组,而且心肌特异蛋白也显著低于对照组,MHC6蛋白表达延后。结论TNNI3K基因能增强心肌细胞的生成,促进m ESC向心肌细胞的分化。

Aim To study the effect of TNNI3 K gene on differentiation of mouse embryonic stem cells into cardiomyocytes.Methods Firstly,we identified the omnipotency of m ESC by the morphological characteristics and cellular immunofluorescence,alkaline phosphatase test（ ALP） and HE staining.In addition,Embryonic body,cultured by hanging drop method,differentiated spontaneously into beating cardiomyocytes,which were identified by cellular immunofluorescence and transmission electron microscopy（ TEM）.Furthermore,h TNNI3 K gene and siRNA,carried by Lentivirus,infected m ESC more than 10 days,respectively,and spontaneously differentiated into mature cardiomyocytes,the differences of myocardial marker proteins expression levels were ananlyzed by flow cytometry（ FCM）,cellular immunofluorescence,Western blot.Results The membrane proteins of m ESC,SSEA-1 and Oct-4,were presented green fluorescence,and ALP test presented blue-purple as well as HE staining with karyoplasmic ratio 1.What＇s more,it was cTnⅠ,cTnT,MLC2 and α-actinin that were clearly visible by cellular immunofluorescence,as well as the unique structure of the muscle fibers by TEM.Furthermore,the cTnT＋positive cells rate of TNNI3K-overexpression group was remarkably higher than that in control group,likely the expression of cTnⅠ,MLC2,GATA4,et al.Interestingly,the contraction protein MHC6 expressed earlier than other groups.The rate of cTnT＋positive cells in the siRNA group was significantly lower than that of the control group,the same as the expression of other cardiac specific proteins.Conclusions TNNI3 K gene might enhance the synthesis of cardiomyocytes and promote the differentiation of m ESC into cardiomyocytes.