差速贴壁法纯化分离GFP转基因小鼠骨髓间充质干细胞

Isolation and Purification of BMSCs of GFP Transgenic Mouse Using the Method of Adhering to Cuture Plastic in Different Time

目的运用差速贴壁法分离纯化绿色荧光蛋白(GFP)转基因小鼠骨髓间充质干细胞(BMSCs)。方法分离GFP转基因小鼠骨髓细胞直接种植在培养瓶中,分别于2h、4h、6h、8h、10h、12h、14h、16h、24h和48h进行首次全量换液,培养3d后按细胞类型对贴壁细胞分别计数,并用CD44、CD45、CD54进行免疫细胞化学染色。另外,选4h、8h、24h后换液的细胞进行传代培养,传至第5代,计算扩增倍数,用CD44、CD45、CD54进行免疫细胞化学染色。结果随着首次换液时间的延长,贴壁细胞密度增多,BMSCs的比例减少。首次换液时间在接种后2h的细胞纯度高,但细胞密度低;24h后的细胞密度高而纯度低;8h后的细胞密度大具有较高的纯度。传代后的GFP转基因小鼠BMSCs能稳定表达GFP。结论差速贴壁法能有效分离纯化GFP转基因小鼠BMSCs,首次换液时间在接种后8～10h时的传代细胞纯度高,具有扩增能力,可作为组织工程和基因治疗研究的种子细胞。

Objective To adopt the method of adhering to culture plastic in different time for cultivating and purifying BMSCs of green fluorescent protein（GFP） transgenic mice. Methods Bone marrow cells isolated from GFP transgenic mice are directly planted in culture flask and an exchange of the total volume medium is made at different time. Then the cells adhering to culture plastic are differently counted according to the ceil types and are examined by immunohistochemistry using the antibodies of CD44, CD45 and CD54 in three days. Moreover, the cells after the exchange of the total volume medium in 4 hours, 8 hours and 24 hours are selected and successively subcultured down to the fifth passage. Then the result of amplification is calculated and the cells are examined by immunohistochemistry using the antibodies of CD44, CD45 and CD54. Results With the extending of the time for the first exchange of medium, the density of cells adhering to culture plastic increased accordingly, but the BMSCs proportion decreased. The cells after first exchange of medium in 4 hours had high BMSCs proportion but low BMSCs density, and the cells in 24 hours had high BMSCs density and low BMSCs proportion. However, the cells in 8-10 hours had high BMSCs density and also high BMSCs proportion. The subcultured BMSCs could stably express GFP. Conclusion The method of adhering to culture plastic in different time for cultivating and purifying BMSCs of GFP transgenic mice is effective. It is suitable to make the first exchange of total volume medium in 8-10 hours. The subcuhured cell has the capacity for amplification and will probably be a seed cell for the research of tissue engineering and gene therapy.