[Isolation, in vitro culture and identification of cardiac stem cells from neonatal SD rats].
[Isolation, in vitro culture and identification of cardiac stem cells from neonatal SD rats].
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发表时间：
2006-11
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通讯作者：
Dong-feng Lu;Hao Wu;Jing-Shuang Huang;Yan Li
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作者：
Dong-feng Lu;Hao Wu;Jing-Shuang Huang;Yan Li
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。结果心肌干细胞的体外培养条件为：1.方法取新生SD大鼠心肌组织，切成0.5- 1.0mm（3）的小块，用0.2%胰蛋白酶和0.1% Ⅱ型胶原酶在37 ℃消化5 min，消化2次。剩余组织在37 ℃、5% CO（2）存在下在完全外植体培养基（CEM）中培养。大约一周后，从贴壁外植体中产生了一层成纤维细胞样细胞。将这些细胞传代，并以约1x 10（6）个细胞/ml接种于含有心球生长培养基的聚-D-赖氨酸包被的多孔板中。当观察到培养细胞搏动时（在第2周），进行流式细胞术和免疫组织化学以鉴定原代和传代细胞。结果消化心肌组织成功培养出原代细胞，流式细胞仪检测细胞表型为c-kit（+）CD 31（+）CD 34（-）CD 45（-）CTnT（-）。细胞传代约2周后，镜下可见单个搏动细胞和同步收缩的细胞团，其表型转化为c-kit（+）CD 31（-）CD 34（-）CD 45（-）CTnT（+）。免疫组化染色证实CTnT在传代细胞中表达，但在原代细胞中不表达。结论从新生SD大鼠心脏组织中获得了c-kit（+）CD 31（+）CD 34（-）CD 45（-）CTnT（-）细胞群，该细胞群具有体外分化为搏动心肌细胞和表达CTnT蛋白的潜能。
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 min at 37 degrees C; with 0.2% trypsin and 0.1% collagenase II. The remaining tissues were cultured in complete explant culture medium (CEM) at 37 degrees C; in the presence of 5% CO(2). About a week later, a layer of fibroblast-like cells was generated from the adherent explants. These cells were passaged and seeded at about 1x10(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.