血管内皮生长因子对诱导多能干细胞向心肌细胞分化的影响

The effect of vascular endothelial growth factor on differentiation of mouse induced pluripotent stem cells into cardiomyocytes

目的观察血管内皮生长因子(VEGF)对小鼠诱导多能干细胞(iPSCs)向心肌细胞分化的诱导作用。方法采用悬滴培养法,分别以10、20、50 ng/ml的VEGF对iPSCs细胞进行诱导分化,自然分化作为阴性对照组,加入1%二甲亚砜诱导剂为阳性对照组,倒置显微镜下观察细胞生长情况,记录跳动的拟胚体出现的时间和数目,计算心肌细胞分化率,细胞免疫荧光检测心肌细胞cTnT的表达,RT-PCR检测心肌发育基因α-MHC和β-MHC mRNA的表达。结果与自然分化组相比,三个浓度组的VEGF均可提高iPSCs的心肌细胞分化率(P<0.05),浓度为20 ng/ml时,iPSCs的心肌细胞分化率最高;与二甲亚砜组相比无统计学差异(P>0.05)。分化的心肌细胞可自发搏动,同时表达心肌特异蛋白cTnT和调控心肌发育的基因α-MHC和β-MHC;VEGF可上调α-MHC和β-MHC的表达(P<0.05)。结论 VEGF可以促进诱导iPSCs向心肌细胞分化。

Objective To observe the effect of vascular endothelial growth factor（VEGF） on differentiation of mouse induced pluripotent stem cells（iPSCs） into cardiomyocytes.Methods Hanging drop method was used to make iPSCs start differentiating.10,20,50 ng/ml VEGF were respectively added to induce the differentiation of iPSCs.Natural differentiation without any inducer was negative group and 1% dimethyl sulfoxide（DMSO） was added as positive control group.Cells were observed under inverted microscope,the time of beating embryonic bodies（EBs） appeared and numbers were recorded and the differentiation rate of myocardial cells was calculated.cTnT expression of myocardial cells was detected by immunofluorescence and α-MHC and β-MHC mRNA expression of myocardial proteins were detected by RT-PCR.Results Compared with the natural differentiation group,three concentrations of VEGF all improved differentiation rate of myocardial cells（P〈0.05）,20 ng/ml VEGF had the most significant effect,which had a rate of 53.17% ± 3.63% and had no significant difference compared to dimethyl sulfoxide group（P〈0.05）.Myocardial cells derived from iPSCs beated spontaneously and expressed cardiac-specific protein cTnT and cardiac genesα-MHC and β-MHC.VEGF upregulated α-MHC and β-MHC expression compared to natural differentiation group（P〈0.05）.Conclusions VEGF can promote the differentiation of pluripotent stem cells into cardiomyocytes.