兔口腔黏膜固有层多能干细胞的分离培养

Isolation and culture of multipotent stem cells from rabbit mucosa lamina propria

目的:探讨兔口腔黏膜固有层中是否含有间充质干细胞并对其进行分离培养。方法:利用间充质干细胞的粘附特性,对兔口腔黏膜固有层多能干细胞进行筛选分离。体外培养扩增后,以CD44、角蛋白抗体(Ck)、波形丝蛋白(Vim)抗体,免疫组化方法对细胞进行鉴定;并用MTT方法检测其增殖,流式细胞术检测其细胞周期。结果:所筛选培养的细胞可在体外呈克隆性生长,细胞表面表达CD44分子,而不表达Ck、Vim;细胞增殖活力旺盛;流式细胞检测结果显示95.1%的细胞处于G0/G1期,与未经筛选的对照组有明显差异。结论:本实验证实在兔口腔黏膜固有层中含有成体干细胞,这类细胞可在体外成功分离并进行扩增培养。口腔黏膜固有层可能是间充质干细胞的又一重要来源,为研究间充质干细胞和口腔黏膜的生物学特性提供基础。

Objective： To isolate multipotent stem cells from rabbit oral mucosa lamina propria and expand them in vitro. Method： Rabbit mucosa lamina propria were digested with collagenase. The isolated cells were cultured for 6h in vitro. And the adherent cells were harvested and expanded in vitro. Then the cells were identificated by immunohistochemistry, used antibody of CD44, cytokeratin and vimentin. The proliferation activity of the selected cells was tested by MTT. Furthermore, the cell cycle was analyzed by flow cytometer （FCM）. Result; The selected cells had behaved like clonogenic ceils and showed high activity of proliferation in vitro. And expression of CD44 on these cell surface was positive, cytokeratin and vimentin were negative. The result of FCM showed that 95.196 of cells lied in G0/G1 period of cell cycle. There was a significant difference between the tested group and control group. Conclusion： Adult stem cells exist in rabbit oral mucosa lamina propria. And the kind of cells can be isolated and cultured in vitro. Oral mucosa lamina propria may be another source of adult mesenchymal stem cells.