摘要
目的建立从Lewis大鼠骨髓中分离、培养间充质干细胞的方法,并对分离、培养的间充质干细胞进行鉴定.方法采用Percoll(1.073g/L)密度梯度分离和贴壁培养相结合的方法分离培养Lewis大鼠的骨髓间充质干细胞(MSC).MSC细胞鉴定:采用相差显微镜观察MSC的形态学特征;流式细胞仪检测细胞表面标志抗原CD29,CD90,CD34和CD45的表达率;苏丹Ⅳ染色检测MSC成脂细胞分化;碱性磷酸酶染色和茜素红染色检测MSC成骨分化.结果原代培养的MSC于24 h后贴壁,48～72 h形成集落,12～15 d可达到80%～90%融合.第1代细胞表面标记物CD29,CD90,CD34和CD45的阳性率分别为80.41%,66.27%,2.73%,0.74%.第3代细胞表面标记物CD29,CD90,CD34和CD45的阳性率分别为89.91%,88.17%,0.81%,0.17%.细胞周期分析显示,第5代细胞G0/G1期细胞占68.9%,S+G2+M期为31.1%.用α-MEM培养液培养的第6代细胞部分分化为成骨和成脂细胞.结论采用Percoll(1.073g/L)密度梯度分离和贴壁培养相结合的方法能够成功分离和培养大鼠的MSC.第5代MSC细胞保持分化潜能.
Objective To establish a method for isolation and culture of bone marrow mesenchymal stem cells（MSC） from Lewis rat in vitro and to identify characteristic of the ceils after culture expansion. Methods MSC were isolated and cultivated from the bone marrow of Lewis rats by Percoll gradient centrifugation and investigated their adherence-dependent growth characters. The morphology of MSC was observed under phase contrast microscope. The expression of CD90, CD45, CD34 and CD29 of the cells were analyzed by flow cytometry. Adipogenic differentiation was studied by Sudan IV staining, osteogenic differentiation by alkaline phosphatase（AP） staining, bone nodule formation by Alizarin red S staining, and cell cycle by flow cytometry. Results After 24 hours primary culture, MSC adhered to plastic surface of the culture dish. After 48-72 hours culture, the ceils proliferated in colonies. These primary MSC reached 80 %-90 % of confluence in 12-15 days. The positive rates of CD29, CD90, CD34, and CD45 in MSC at P1 （first generation） were 80.41%, 66.27%, 2.73%, and 0.74% respectively, and at P3 89.91%, 88.17%, 0.84%, and 0.17% respectively. Cell cycle studies revealed that while a small fraction of MSC at Ps （fifth generation） are actively engaged in proliferation（approximately 31.1%at S ＋ G2 ＋ M）, the vast majority of ceils are standing at the G0/Gx phase of the cell cycle （approximately 68.9 % at G0/G1 ）. Part of culture-expanded MSC at P6 （sixth generation） cultivated in a-minimum essential medium（a-MEM） showed positive by alkaline phosphatase staining, Alizarin red staining, and Sudan Ⅳ staining. Conclusion Mesenchymal stem ceils can be successfully isolate and cultivate from rat bone marrow by Percoll gradient centrifugation and keeps its adherence-dependent growth characters. They retain muhipotentiality after culture expansion at P5.
出处
《福建医科大学学报》
2005年第4期360-363,共4页
Journal of Fujian Medical University
基金
福建省教育厅科研基金资助项目(JB04211)
