新生小鼠心肌干细胞培养与分化的研究

Study on Cultivation and Differentiation of Cardiac Stem Cells from Neonatal Mice

目的了解新生小鼠心肌干细胞原代和传代的培养及其分化潜能,为缺血及坏死心肌重建的临床应用提供科学依据。方法在无菌超净台中剪取新生小鼠心脏组织,经胰酶反复消化后,弃去消化液,所得剩余组织块用胶头滴管反复吹打后离心,加入培养液培养,待其长满培养瓶后进行传代,传代培养约1周可见搏动的心肌细胞,也可见成团细胞的同步收缩。结果采用改良后的方法从消化后的心肌组织块中成功培养得到原代细胞,进行免疫荧光染色、流式细胞仪鉴定其表型为c-kil、8ca-1阳性的细胞,表面不表达CD 34、CD 8、肌球蛋白重链(MHCⅡ)。细胞经传代培养1周左右,开始出现搏动,也可见成团细胞的同步收缩。再次经免疫荧光染色、流式细胞仪鉴定其表型为c-kit、sca- 1阴性,MHCⅡ阳性的细胞,仍然不表达CD 34、CD 8。结论在原方法的基础上,通过对其进行改良,从心脏中成功分离得到纯度更高、活性更好的心肌干细胞,其表型为c-kit^+、sca-1^+、CD 34^-、CD 8^-、MHCⅡ^-,经过传代培养之后,分化为搏动的心肌细胞,细胞表面标记变为c-kil^-、sca-1^-、CD 34^-、CD 8^-,并表达心肌结构蛋白MHCⅡ。

Objective To investigate the potential of primary and passaged cardiac stem cells from neonatal mice in cultivation and differentiation, and provide a scientific basis for the reconstruction of the clinical application to myocardial ischemia and necrosis. Methods Myocardial tissues obtained from neonatal mice were cut into pieces, and digested repeatedly with trypsin. Discard the digestive liquid and leave the remaining tissues. After terminating digestion , use the dropper to beat upon the remaining tissues and centrifuge the myocardial tissues. The remaining tissues were cultured in complete explant culture medium （CEM）. When the cells covered culture-flask, we passage it . after about one week , not only we can see the beating myocardial cell, but also can see the cells synchronized contraction. Results We use the improved method to digest and culture the myocardial tissues , and get the better results than the original method. Flow cytometry and Immunofluorescence were performed for identification of the primary and passaged cells. The primary cells were successfully cultured from the digested myocardial tissue, both flow cytometry and immunofluorescence demonstrated the phenotype of c- kit^＋,sca- 1^＋ , CD 8^- , CD 34 - , MHC Ⅱ - After cell passage for about one week, single beating cells and cell clusters with synchronized contraction were seen microscopically, and their phenotype was converted to c-kit^- , sca- 1^ - , CD 8^ - , CD 34^ - , MHC Ⅱ^ ＋. Conclusion On the basis of the original method, we use the improved method and succeed to get higher purity and better activity of cardiac stem cells. The phenotype of primary cells are c- kit^＋ , sca- 1 ^＋ , CD 34^ - CD 8^ - , and MHC Ⅱ ^-. After passaged culture, cardiac stem cells change into beating cardiac myocyte which phenotype are c-kit^＋ , sca-1^ -, CD 34^- , CD 8^- , and the myocardial structural protein （MHC Ⅱ） also express on cardiomyocyte.