L-抗坏血酸促进人胚胎干细胞向心肌细胞分化的研究

Effect of L-ascorbic acid on differentiation of human embryonic stem cells into cardiomyocytes in vitro

目的用不同浓度的L-抗坏血酸(VitC)作为分化基础培养液中的诱导剂,观察其对人胚胎干细胞(hESC)分化为心肌细胞的影响,探寻一种成分明确、成本低廉且高效的诱导方法。方法复苏hESC,使用干细胞培养液培养至细胞融合度90%后,分别用含不同浓度(0、10、20、30、40、50、60、70μmol/L)VitC的分化培养液对其进行诱导分化,在光学显微镜下观察分化过程。用实时定量聚合酶链式反应(real-time PCR)分别检测干细胞特异性标志物Nanog和性别决定因子同源盒2(Sox2),心肌细胞特异性标志物心肌肌钙蛋白T(cTnT)、心脏特异性同源盒转录因子(Nkx2.5)的表达。用免疫荧光检测Nanog、cTnT、Nkx2.5的蛋白表达变化。结果在分化基础培养液中,低浓度VitC(10~30μmol/L)不能诱导h ESC向心肌分化,随着VitC浓度的增高,干细胞逐渐往心肌方向分化,心肌分化效率逐渐提高,Nanog和Sox2的表达量逐渐下降;VitC浓度为40μmol/L或50μmol/L组在第8天开始出现自发跳动细胞,且有cTnT和Nkx2.5阳性表达。realtime PCR结果显示随分化时间延长,cTnT和Nkx2.5的mRNA表达逐渐增加,心肌表面标志物Nkx2.5、cTnT的mRNA表达量在VitC浓度40μmol/L和50μmol/L要比60μmol/L和70μmol/L的诱导效果更高。提示浓度大于60μmol/L就开始出现抑制分化的作用。结论实验研究证实VitC在hESC向心肌分化中是不可或缺的因子,研究还明确了VitC诱导h ESC分化为心肌细胞的适宜浓度范围。

Objective To observe the effects of different concentrations of L-ascorbic acid(VitC)as an inducer in differentiation medium on differentiation of human embryonic stem cells(hESC)into cardiomyocytes,and explore the method with clear composition,low cost and high efficiency.Methods The hESC were resuscitated and cultured with stem cell culture medium to 90%cell fusion,and induced to differentiate with differentiation medium containing different concentrations VitC(0,10,20,30,40,50,60,70μmol/L),and differentiation process was observed under optical microscope.The expression of stem cell specific markers Nanog and Sox2,cardiomyocyte specific markers cTnT and Nkx2.5 were detected by real-time quantitative polymerase chain reaction(real-time PCR),and the protein expression of Nanog,cTnT and Nkx2.5 was detected by immunofluorescence.Results In differentiation medium,low concentration of VitC(10-30μmol/L)could not induce hESC to differentiate into myocardium.With the increase of VitC concentration,the efficiency of hESC differentiating into cardiomyocyte,myocardium was gradually improved,and the expression of Nanog and Sox2 gradually decreased.On the 8th day,spontaneous beating cells appeared in VitC concentration with 40μmol/L or 50μmol/L group,and cTnT and Nkx2.5 were positively expressed.The results of real-time PCR showed that mRNA expression of cTnT and Nkx2.5 gradually increased with prolongation of differentiation time.The mRNA expression of myocardial surface markers Nkx2.5 and cTnT with 40μmol/L and 50μmol/L concentration was higher than that with 60μmol/L and 70μmol/L concentration,suggesting that the concentration greater than 60μmol/L inhibited differentiation,and the effect of inhibiting differentiation began to appear when concentration was greater than 60μmol/L.Conclusion It is demonstrated that VitC is an indispensable factor in differentiating hESC into cardiomyocytes,and also clarifies the appropriate concentration range of VitC which could induce hESC to differentiate into cardiomyocytes.