安徽医科大学学报2013，Vol.48Issue(6)：607-611,5.
大鼠骨髓间充质干细胞体外培养及CM-DiI标记的实验研究
Study on cultivation and CM-DiI labeling of rat bone mesenchymal stem cells in vitro
摘要
Abstract
Objective To establish a method for isolation, purification, proliferation and labeling of bone marrow mwsenehymal stem cells ( MSCs )in vitro. Methods MSCs were isolated and purified by whole bone marrow adherence and differential adhesion separation. The morphological characteristics were observed in incubation period. Cell cycle and surface makers were analyzed by flow cytometry as well as induction of adipogenic and osteogenic differentiation for identification of MSCs. MSCs were labeled with chloromethyl-benzamidodialkylarbocyanine( CM-DiI), observed by fluorescence microscope and analyzed by flow cytometry. The impact of CM-DiI on growth and proliferation of MSCs were recorded by drawing growth curve. Results MSCs mostly showed circular, polygonal, spindle after inoculation for 24 ~48 h. At the third passage, cells formed a uniform long spindle shape. MSCs at passage 3 were strongly positive for CD44, weakly positive for CD106, but negative for CD11b and CD45. There were more than 85% of MSCs in the G0/G1 phase. After adipogenic and osteogenic induction, oil red O and alizarin red staining were positive respectively. CM-Dil labeled cells showed red fluorescence under fluorescence microscope. Florescence emerged by CM-DiI was not significantly weakened after labeled 2 weeks in vitro. Cell labeling rate was up to 95% . CM-DiI did not effect on cell growth and proliferation. Conclusion A great number of MSCs with high purity have been obtained by method of whole bone marrow adherence and amplification. The tracing method of CM-DiI labeling cells is simple, stable, and non-toxic.关键词
骨髓间充质干细胞/原代培养/CM-DiI/细胞标记Key words
MSCs/ primary culture/ CM-DiI/ cell labeling分类
医药卫生引用本文复制引用
毛雨红,莫碧文,李劳冬,李洁,王孝养,韦江红,黄剑伟,徐青..大鼠骨髓间充质干细胞体外培养及CM-DiI标记的实验研究[J].安徽医科大学学报,2013,48(6):607-611,5.基金项目
国家自然科学基金(编号:30760083) （编号:30760083）
广西自然科学基金重点课题(编号:2012GXNSFDA053020) （编号:2012GXNSFDA053020）
广西科学研究与技术开发计划课题(编号:0719006-2-8) （编号:0719006-2-8）
广西卫生厅重点课题(编号:200732200976) （编号:200732200976）
广西青年科学基金(编号:0991085) （编号:0991085）
