心肌干细胞培养和纯化的实验方法研究  

A Study on the Experiment Method for Cardiac Stem Cells Culture and Purification

作　　者：姜宏[1] 施广飞[2] 

机构地区：[1]南京中医药大学第一临床医学院,江苏南京210046 [2]南京大学医学院附属鼓楼医院心血管科,江苏南京210008

出　　处：《现代生物医学进展》2009年第1期33-35,49,共4页Progress in Modern Biomedicine

基　　金：江苏省卫生厅科研项目(H200220);南京市卫生局科研项目(ZKX0206)

摘　　要：目的:探讨心肌干细胞(CSC)体外分离、培养和纯化方法,以建立重复性好、稳定性高的CSC培养的方法。方法:用不同的消化方法来分C57BL小鼠心脏组织中的细胞,将获得的细胞置于自行拟定的心肌干细胞培养液中培养。以c-kit、Sca-1和Sca-1/c-kit作为干细胞标记,用免疫磁珠法纯化细胞。用流式细胞仪、免疫荧光显微镜、激光共聚焦显微镜检测分选后所得细胞表面标记表达,并对比观察分选前后细胞生长情况,进一步了解CSC的生物学特性。结果:①从小鼠心肌组织中分离培养所得的细胞中,含有一种类似于文献报道中所提及CSC形态的细胞。②用免疫磁珠法可以分选出高纯度的CSC,实验中分选出CSC分别具有c-kit+-CD34--lin-、Sca-1+-CD34low-Lin-、Sca-1+-c-kit+-CD34low-Lin-的表型。③分选后的干细胞经过1～2d的滞留期后,贴壁生长的细胞形态相对均一;而传代后细胞贴壁迅速,细胞形态多样。结论:从C57BL小鼠心脏中可以分离、培养获得CSC,并可用免疫磁珠法得以纯化。Objective： To explore the conditions of isolation, culture and purification of Cardiac stem cells （CSC） in vitro, and to find the best method of dissociating and culturing the CSC reproducibly and stably. Methods： The cells in the C57BL mice heart were dissociated and extracted by different ways, then the acquired cells were put in the CSC culture fluid made by ourselves, c-kit, Sca-1 and Sca-1/c-kit were used as CSC markers for purification by sorting with the magnetic activated cell sorting system. CSC markers were identified by flow cytometry, immunofluorescence microscope and laser confocal microscopy. The cells＇ growth status before and after purification were observed and studied contrastly for further understanding of the CSC biologic features. Results： （1）A kind of cell whose morphology was similar to the CSC mentioned in the literature was dissociated and cultured from C57BL mice heart in vitro. （2） Highly purified CSC were achieved by immunomagnetic beads. These CSC had phenotypes of c-kit^＋ -CD34^- -lin^-, Sca-1^＋ -CD34^low -Lin^- and Sca-1^＋ -c-kit^＋ - CD34^low -Lin^-. （3） After purification, the CSC which kept staying in the culture for 1-2d detention period, began growing adherence with relatively uniform morphology, whereas the speed of cell adherence after serial subcultivation was faster and the morphology became polymorphous. Conclusion： CSC in the heart of C57BL mice can be dissociated and cultured. It also can be purified by sorting with the magnetic activated cell sorting system.

关 键 词：心肌干细胞 细胞培养 纯化 

分 类 号：Q813[生物学—生物工程] R542.22[医药卫生—心血管疾病]