大鼠心肌干细胞的体外分离培养  

The Isolation,Culture and Identification of Cardiac Stem Cells in Vitro from Neonatal SD Rat

作　　者：吴昊[1] 陆东风[1] 黄璟[1] 李妍[1] 

机构地区：[1]广州医学院第二附属医院心内科,广州510620

出　　处：《血栓与止血学》2006年第5期201-205,共5页Chinese Journal of Thrombosis and Hemostasis

摘　　要：目的自大鼠心脏中分离得到心肌干细胞,体外培养并观察其向心肌细胞的分化能力。方法无菌操作取下新生鼠心脏,经反复消化后,弃去消化液,将所得残余组织块进行培养,视生长情况进行传代(一般1周左右)。传代细胞采用不同培养液进行培养,约2周后,镜下可见搏动细胞时,分别对原代细胞和传代细胞进行流式细胞鉴定及免疫组化染色。结果从消化后残留心肌组织块中成功培养出原代细胞,经流式细胞仪鉴定其表型为c-kit,CD 31阳性,CD 34,CD 45、心肌钙蛋白T(Cardiac Troponin T,CTnT)阴性。细胞经传代培养2周后,单个细胞开始出现搏动,亦可见成团细胞的同步收缩。再次流式鉴定的结果,表型有所改变,主要是CD 31转阴性,CTnT阳性,而c-kit、CD 34、CD 45无明显变化。免疫组化证实,原代细胞并不表达心肌细胞结构蛋白CTnT,而在部分传代细胞内可见CTnT表达。结论成功自心脏中分离得到一类表型为c-kit^+、CD 31^+、CD 34^-、CD 45^-、CTnT^-的细胞,经使用不同成份培养液传代培养,可分化可搏动的心肌细胞,并表达心肌结构蛋白。Objective To find an effective, mature method which can also be repeated easily to isolate and culture CSCs in vitro, and then made them differentiate into beating cardiomyocytes. Methods Isolated myocardial tissue was get from Neonatal rat, and then was cut into 0.5-1 mm^3pieces, digested trice for 5 rain at 37 ℃ with 0.2% trypsin and 0.1% collagenase Ⅱ respectively. The remaining tissue fragments, were cultured as explants in CEM at 37 ℃ and 5% CO2. After about a week, a layer of fibroblast-like cells was generated from adherent explants. These cells were passaged and were seeded at about 1 × 10^6 cells/ml in poly- D-lysine-coated multi-well plates in cardiosphere-growing medium. Both flow cytometry and immunohistochemistry were used for the primary cells and passaged cells which were cultured about 2 weeks and started to beat. Results Cultured the primary cells successfully from the remaining tissue fragments, and the flow cytometry indicated that their phenotype were c-kit^＋ , CD 31^ ＋ , CD 34^- , CD 45^- , CTnT^-. The cells were passaged and cultured in cardiosphere-growing medium. After about 2 weeks, single beating cell and cell-sphere synchronous contraction can be seen through telescope. And their phenotype changed into c-kit^ ＋ , CD 31^ -, CD 34^- , CD 45^-, CTnT^ ＋. Immunohistochemistry stain proved that CTnT exist in the passaged ceils while not in the primary cells. Conclusions Isolated a kind of cells which phenotype were c-kit ^＋ , CD 31^ ＋ , CD 34^-, CD 45^ - , CTnT^-. In vitro they can differentiate into beating cardiac myocyte and express CTnT, specially exist in cardiomyocyte.

关 键 词：心肌于细胞 分离和培养 流式细胞术 免疫组化 

分 类 号：Q25[生物学—细胞生物学]