大鼠原代小胶质细胞的分离和培养方法的改进

A improved method for cultivating primary microglia of the rat

目的:探索改进大鼠原代小胶质细胞培养分离纯化方法,以获取数量多、功能活性稳定的小胶质细胞。方法:在Miriam Mecha的混合胶质细胞培养方法的基础上稍加改良,采用直立手摇法进行分离纯化小胶质细胞,细胞铺板后再采用差速贴壁法进一步得到更纯的小胶质细胞。使用台盼蓝法进行细胞计数;采用CD11b免疫荧光方法对纯化的小胶质细胞进行纯度鉴定;采用原代小胶质细胞吞碳粒实验对其功能活性进行检测。结果:改进的方法可以稳定地获取大约1×106个/培养瓶(75 cm2,10 ml)的小胶质细胞,纯度达到98%,并且其吞噬活性也非常强。结论:改进的原代小胶质细胞培养方法操作时间短,获得细胞数量多、稳定性好、纯度高、功能活性强,且可第二次收获细胞,对于离体条件下进行小胶质细胞的研究具有广泛的实用价值。

Objective： To explore an improved method for cultivating,isolating and purifying primary microglia of the rat,and to gain a significant amount of the microglia with functional and active stability. Methods： The isolate and purify the microglia by shaking the upright flask was slightly improved from the Miriam Mecha＇s cultivating method of the mixed glial cells. After the cells were planked,pure microglia were obtained by differential attachment. At last,the quantity of the primary microglia was counted by the trypan-blue injection method. The purity was identified by the CD11 b immunefluorescence method,and the functional activity was detected by phagocytizing carbon particles. Results： By the improved method,microglia with the density around 1 × 10^6 per flask（ 75 cm^2,10 ml） was steadily obtained with the purity over98%,meanwhile with powerful phagocytic activity. Conclusion： The improved method of microglia cultivating has the following advantages,shorter operation time,larger amount,better stability and power functional activity. Moreover,the cells can be harvested for the second time. Our method will be helpful in the in vitro research of the microglia.