成年小鼠心脏c-kit+干细胞的分离及诱导分化

摘要

Objective To isolate the c-kit + stem cells from the hearts of adult mice,and to explore the induced differentiation of cardiac stem cells (CSC).Methods The freshly isolated hearts were obtained from 3 male C57BL/6 mice,and the hearts were dissected into single cells.The c-kit + stem cells were isolated from the CD45-cells by magnetic microbeads.Flow cytometric analysis and Immunofluorescence were used to measure the purity and the phenotype of c-kit + cells.The cell purity was determined and cell phenotype was observed.The c-kit + cells were cultured in suspension medi um to form the CardioStem sphere.RT-PCR was used to analyze the expression of the smooth muscle cells marker (α-SMA and sm-22α),endothelial cells marker (vWF),myocardial cells marker (cTnT) and cardiac stem cells marker (c-kit) after differentiation.Immunofluorescence was used to measure the phenotypes after the differentiation.Results The purity of c-kit + reached 89.3％,and the cardiac stem cells expressed the c-kit antibody.The primary cells grew slowly,but faster after being passaged.When cultured in suspension,the cells can form the CardioStem sphere.The expression of α-SMA,sm-22α,vWF,and cTnT mRNA was significantly increased,but the expression of c-kit mRNA was significantly decreased on day 14 as compared with that on day 0 after differentiation (all P ＜ 0.05).cTnT-positive cells were observed in cardiomyocyte-induced differentiation medium for about 14 days in cardiac c-kit + stem cells.vWF positive cells and α-SMA positive cells were observed in the medium without LIF on day 14.Conclusion Highly purified c-kit + CSCs can be isolated and cultured from the hearts of adult C57BL mice,c-kit + CSCs can be differentiated into cardiomyocytes,smooth muscle cells,and endothelial cells.%目的 从成年小鼠心脏中分离、纯化c-kit+干细胞,并观察其诱导分化结果.方法 取C57BL/6雄性小鼠3只,取心脏组织,将心脏解离成单个细胞.采用CD45磁珠、c-kit磁珠分选得到c-kit+干细胞.鉴定细胞纯度,观察细胞表型.观察所得心脏c-kit+干细胞球的形成情况,对其进行诱导分化,采用RT-PCR法观察诱导分化细胞血管平滑肌细胞的标志物α-SMA和sm-22α、血管内皮细胞标志物vWF、心肌细胞标志物cTnT、心脏干细胞标志物c-kit,观察诱导分化的细胞表型.结果 心脏c-kit+干细胞c-kit+纯度为89.3％,心脏干细胞表面表达c-kit抗体.原代c-kit+干细胞生长缓慢,传代后细胞生长较快,悬浮培养可形成心脏c-kit+干细胞球.与诱导第0天比较,诱导分化第14 d时心脏干细胞α-SMA、sm-22α、vWF、cTnT mRNA相对表达量升高,c-kit mRNA相对表达量降低,P均＜0.05.心脏c-kit+干细胞在心肌细胞诱导分化培养基中培养14 d左右可见cTnT阳性细胞.心脏干细胞在无LIF的培养基中培养14天可见vWF、α-SMA阳性细胞.结论 成功从成年小鼠心脏分离心脏c-kit+干细胞,所得c-kit+干细胞c-kit+纯度高,可成功诱导分化为心肌细胞、血管平滑肌细胞及血管内皮细胞.