外胚间充质干细胞的体外分离培养与鉴定

背景：外胚间充质干细胞不仅可以作为组织工程研究的一种新种子细胞，而且可以为临床神经系统肿瘤提供一种新的治疗策略。目的：体外分离外胚间充质干细胞，对其进行磁珠筛选、形态及超微结构观察、神经干细胞表型鉴定及生长增殖能力检测。方法：从SD胎鼠的上下颌突分离出外胚间充质干细胞进行原代培养，培养液中加入磁珠包被的低亲和力神经生长因子受体抗体，进行磁珠筛选纯化。结果与结论：经磁珠筛选后的外胚间充质干细胞呈“成纤维细胞样”细胞，为长梭形，漩涡状生长：透射电子显微镜显示主要为间充质样细胞，形状小规则，细胞增殖活性高，处于未分化状念；免疫组织化学染色结果显示人自然杀伤细胞1、波形蛋白、S-100在细胞浆中呈阳性表达，而细胞角蛋白则为阴性；生长曲线显示呈“S”形，第3天进入对数生长期，第6天进入平台期，其倍增时问TD=40．28h。提示体外分离经磁珠筛选纯化扩增的细胞证实为外胚间充质干细胞，其具有干细胞特征。

In vitro isolation, cultivation and identification of ecto-mesenchymal stem cells

BACKGROUND： Ecto-mesenchymal stem ceils are not only a new kind of seed cells in tissue engineering research, but also an innovative therapeutic strategy for tumors in clinical nervous system. OBJECTIVE： To observe morphology and ultrastructure, and the proliferation of in vitro isolated ecto-mesenchyma dentify nerve stem cell marker and test the ability of growth stem cells. METHODS： Ecto-mesenchymal stem cells were enzymatically isolated from the first branchial arch of rat embryo and maintained in an undifferentiated state with beads coated low affinity nerve growth factor receptor antibody. Then they were purified by magnetic activated cell sorting. RESULTS AND CONCLUSION： Ecto-mesenchymal stem cells after purification by magnetic activated cell sorting grew like swirls and were fibroblast-like and long spindle-shaped. The ultrastructure of ecto-mesenchymal stem cells was characterized by mesenchymal-like cells with irregular shape, undifferentiation and high activity of proliferation. Immunohistochemical staining manifested that ecto-mesenchymal stem cells expressed human natural killer cells-l, Vimentin and S-100 but not cytokeratin. Ecto-mesenchymai stem cells grew into the exponential phase after cultured for 3 days, and reached to plateau phase after cultured for 6 days. The growth curve was ＂S＂ like and the doubling time was 40.28 hours. The evidence suggested that the ceils in vitro cultured and purified by magnetic activated cell sorting were undifferentiated ecto-mesenchymal stem cells with stem cell characteristics.