CAJ | 학술논문

目的利用维生素C作为诱导因子,采用悬滴法形成拟胚体,体外诱导小鼠胚胎干细胞分化为心肌细胞并检测其分化效率,同时确定这种诱导方法的最佳维生素C浓度.方法复苏小鼠胚胎干细胞,传代培养后,消化离心后重悬细胞,用悬滴法形成拟胚体,用含1×10-3、1×10-4、1×10-5、1×10-6 mol/L 4种不同浓度维生素C的分化培养基对其进行诱导分化,以不添加任何诱导剂作为对照组,观察各组小鼠出现跳动拟胚体的数量,并计算分化效率;免疫荧光染色检测心肌细胞特异标志物肌钙蛋白T(cTnT);膜片钳实验检测心肌细胞自发性动作电位.结果大量的自发跳动心肌细胞在诱导分化后12d左右开始出现.维生素C诱导小鼠胚胎干细胞分化为心肌细胞的最佳浓度为1×10-4 mol/L,其分化出现跳动拟胚体的百分比为81.25％,显著高于不加诱导剂的对照组(12.50％);跳动心肌细胞cTnT染色阳性;跳动心肌细胞检测到自发性动作电位.结论最佳的维生素C浓度(1×10-4mol/L)能够明显提高体外悬滴法诱导小鼠胚胎干细胞分化为心肌细胞的效率.
목적 이용유생소C작위유도인자,채용현적법형성의배체,체외유도소서배태간세포분화위심기세포병검측기분화효솔,동시학정저충유도방법적최가유생소C농도.방법 복소소서배태간세포,전대배양후,소화리심후중현세포,용현적법형성의배체,용함1×10-3、1×10-4、1×10-5、1×10-6 mol/L 4충불동농도유생소C적분화배양기대기진행유도분화,이불첨가임하유도제작위대조조,관찰각조소서출현도동의배체적수량,병계산분화효솔;면역형광염색검측심기세포특이표지물기개단백T(cTnT);막편겸실험검측심기세포자발성동작전위.결과 대량적자발도동심기세포재유도분화후12d좌우개시출현.유생소C유도소서배태간세포분화위심기세포적최가농도위1×10-4 mol/L,기분화출현도동의배체적백분비위81.25％,현저고우불가유도제적대조조(12.50％);도동심기세포cTnT염색양성;도동심기세포검측도자발성동작전위.결론 최가적유생소C농도(1×10-4mol/L)능구명현제고체외현적법유도소서배태간세포분화위심기세포적효솔.
Objective Using the vitamin C as inducers and hanging drop method to form embryoid bodies to induce the differentiation of mouse embryonic stem cells into cardiomyocytes in vitro and detecting its differentiation efficiency.Searching for the optimal concentration of vitamin C for this method.Methods Mouse embryonic stem cells were recovered,passaged,digested,centrifuged,suspended and form embryoid bodies by hanging drop method.The embryoid bodies were induced by differentiation medium containing various concentration of vitamin C (1 × 10-3,1 × 10-4,1 × 10-5,1 × 10-6 mol/L).Control group was not treated by inducer.The number of beating embryoid bodies were calculated.Staining the specific marker cardiac Troponin T (cTnT) of cardiomyocytes by immunofluorescence; Detecting the electrophysiological function of cardiomyocytes by patch clamp experiment.Results Widespread spontaneous beating cardiomyocytes was typically observed by 12 d after differentiation.The optimal concentration of vitamin C to promote the cardiac differentiation of mouse embryonic stem cells (mESC) was 1 × 10-4 mol/L,and the percent of beating colonies was 81.25％,significantly higher than 12.50％ of the control group.Beating cardiomyocytes were positive to cTnT staining; Spontaneous action potential of beating cardiomyocytes was detected.Conclusion The optimal concentration of vitamin C (1 × 10-4 mol/L) could significantly promote the cardiac differentiation of mESC by hanging drop method in vitro.