新生SD大鼠心肌干细胞的体外分离培养与鉴定  

Isolation,in vitro culture and identification of cardiac stem cells from neonatal SD rats

作　　者：陆东风[1] 吴昊[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,广东广州510260

出　　处：《南方医科大学学报》2006年第11期1629-1632,共4页Journal of Southern Medical University

摘　　要：目的自新生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分化的能力。方法无菌操作取下新生鼠心脏,经反复消化后,弃去消化液,所得残余组织块进行培养,视生长情况进行传代。传代细胞采用诱导分化培养液进行培养,细胞生长至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、CD31阳性,CD34、CD45阴性,心肌钙蛋白T(CTnT)阴性。细胞经传代培养两周后,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,表型有所改变,主要是CD31转阴性,CTnT阳性,而c-kit、CD34、CD45无明显变化。免疫组化再次证实,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+CD31+CD34-CD45-CTnT-的细胞,经使用不同成分培养液传代培养,可分化为可搏动的心肌细胞,并表达心肌结构蛋白。Objective To isolate and culture cardiac stem cells （CSCs） in vitro and evaluate their potential of differentiation into functional cardiac myocytes. Methods Myocardial tissues obtained from neonatal SD rats were cut into pieces of 0.5-1.0 mm^3, and digested twice for 5 rain at 37 ℃ with 0.2% trypsin and 0.1% collagenase II. The remaining tissues were cultured in complete explant culture medium （CEM） at 37 ℃ in the presence of 5% CO2. About a week later, a layer of fibroblast-like cells was generated from the adherent explants. These cells were passaged and seeded at about 1 ×10^6 cells/ml in poly-D-lysine-coated multi-well plates in cardiosphere-growing medium. When beating of the cultured cells was observed （at week 2）, flow cytometry and immunohistochemistry were performed for identification of the primary and passaged cells. Results The primary cells were successfully cultured from the digested myocardial tissue, and flow cytometry demonstrated the phenotype of c-kit^＋CD31^＋CD34^-CD45^-CTnT^-. After cell passage for about two weeks, single beating cells and cell clusters with synchronized contraction were seen microscopically, and their phenotype was converted to c-kit^＋CD31^＋CD34^-CD45^-CTnT^＋. Immunohistochemistry staining identified CTnT expression in the passaged cells but not in the primary cells. Conclusions A cell population with the phenotype c-kit^＋CD31^＋CD34^-CD45^-CTnT^- has been obtained from neonatal SD rat heart, which possesses the potential to differentiate in vitro into beating cardiac myocytes and express CTnT protein.

关 键 词：心肌干细胞 分离和培养 流式细胞术 免疫组化 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]