一种改良的新生大鼠小胶质细胞原代培养方法

A modified method for neonatal rat microglia primary culture

目的:改良现有的小胶质细胞纯化分离培养方法,建立稳定、高产的培养模型。方法:选用新生1～2d SD大鼠进行混合胶质细胞培养,然后结合营养剥离法及震摇法纯化分离小胶质细胞;利用CD11b/c(OX42)免疫细胞化学的方法对分离的小胶质细胞纯度进行鉴定。结果:改良的方法可稳定的获得5×104个/培养瓶(25cm2)的小胶质细胞,纯度达到95%,荧光倒置显微镜下观察细胞形态,分离第1 d小胶质细胞呈不规则圆形,折光不均匀,继续培养3-5 d,小胶质细胞逐渐出现单极或多极突起,7 d时部分细胞转为静止状态,呈分枝状。结论:改良的小胶质细胞培养方法产量多,纯度高,为小胶质细胞在中枢神经系统疾病相关研究提供了新的基础。

Objective： To modify the current method and establish a stable method to improve primary culture and puri- fication of microglia. Methods ： Mixed glia cultures were prepared from cerebral cortiex of 1 - 2 days old SD rat. The cells were purified with the combined method of nutritional deprivation and shaking methods. Then the purity of isolated cells was identified by expression of CD11 b/c （OX42） using immunocytochemical technique. Results： With the modified method we successfully obtained 5 × 10^4 cells per flask （25 cm2） with high purity （ 〉95% ）. Morphological changes of cultured cells were observed under an inverted phase contrast microscope. At the first day after purification, the adherent microglia cells were rounded with irregular margin. The cells began to have single or muhipole protuberance with uneven refraction after 3-5 d culture. Part of the cells began to change to ramified rest situation after 7 d culture. Conclusion： The modified culture method was effective to increase the production rate and purity of the microglia, which provides a no- vel base for the microglia related central nervous system diseases researches.