乳鼠心脏Sca-1^+干细胞定向分化的诱导研究

Research on directed differentiation of Sca-1 positive stem cells in neonatal mouse hearts

目的研究抗坏血酸(ascorbic acid,AA)即维生素C(vitamin C,Vc)、地塞米松(dexamethasone,DXMS)及促红细胞生成素(erythropoietin,EPO)对Sca-1+干细胞分化为心肌细胞的定向诱导作用。方法急性分离并培养C57BL/6J乳鼠心脏来源的Sca-1+干细胞至第3代或第4代,培养基中分别加入抗坏血酸、地塞米松及EPO,以不同的诱导方案促使其分化,分组如下:空白对照组、DMSO对照组、Vc诱导组、DXMS诱导组、EPO诱导组、Vc及DXMS联合诱导组。干细胞在不同诱导剂作用下持续培养3周,期间利用光镜观察细胞形态改变;第3周时进行Nkx2.5和cTnI免疫荧光染色来判断干细胞的分化效率。结果光镜观察显示,Sca-1+干细胞在抗坏血酸、地塞米松及EPO的诱导下,均有不同程度向心肌细胞分化的形态学变化。免疫荧光染色结果显示,分化细胞中的心肌细胞特异性标记物cTnI及Nkx2.5的阳性率均低于50%,其中cTnI阳性率分别为:空白对照组(9.41±1.67)%、DMSO对照组(10.42±3.20)%、Vc诱导组(14.84±1.99)%、DXMS诱导组(10.68±2.27)%、EPO诱导组(8.64±2.98)%、Vc及DXMS联合诱导组(44.27±22.91)%。与空白对照组相比,仅联合诱导组cTnI表达水平明显上调[(9.41±1.67)%vs(44.27±22.91)%,P<0.05],其余各组与空白对照组相比差异均无统计学意义。Nkx2.5在各组的阳性率较低,Vc及DXMS联合诱导组仅为(0.05±0.02)%,其余各组阳性率均低于该水平。结论抗坏血酸、地塞米松及EPO在Sca-1+干细胞分化为心肌细胞的过程中有一定的诱导作用,其中,抗坏血酸和地塞米松联合诱导组可在较高水平上诱导Sca-1+干细胞分化为心肌细胞。

Objective To investigate the direct induction effects of ascorbic acid(AA or vitamin C,Vc),dexamethasone(DXMS)and erythropoietin(EPO)on the differentiation of Sca-1+stem cells into cardiomyocytes.Methods Sca-1+stem cells from C57BL/6J mouse hearts were acutely isolated and cultured to the 3 rd or 4 th generation.Sca-1+stem cells were treated with different induction protocols of AA,DXMS and EPO to differentiate into cardiomyocytes.The experiment was grouped as follows:blank control group,DMSO control group,Vc group,DXMS group,EPO group,Vc+DXMS group.The stem cells were cultured for 3 weeks under different inducing agents.The morphological changes of stem cells were observed by light microscopy.After 3 weeks of cell culture,the immunofluorescence staining of Nkx2.5 and cTnI was performed to determine the differentiation efficiency of stem cells.Results Under light microscope,there were morphological changes in different degrees of Sca-1+stem cells differentiation to myocardial cells after treated with AA,DXMS and EPO.The immunofluorescence staining results illustrated that the positive rates of cTnI and Nkx2.5 as cardiomyocyte specific markers in different treatment groups were less than 50%.The positive rates of cTnI were(9.41±1.67)%in blank control group,(10.42±3.20)%in DMSO control group,(14.84±1.99)%in Vc group,(10.68±2.27)%in DXMS group,(8.64±2.98)%in EPO group,and(44.27±22.91)%in Vc+DXMS group.The positive rate of cTnI in Vc+DXMS group was significantly higher than that in DMSO group(P<0.05),while there was no statistically significant difference between the other groups and DMSO group.The positive rate of Nkx2.5 was lower in each group,with only(0.05±0.02)%in Vc+DXMS group.Conclusion AA,DXMS and EPO may differentiate Sca-1+stem cells into cardiomyocytes to some extent.Especially,the combination of AA and DXMS can promote Sca-1+stem cells differentiation into cardiomyocytes at a high level.