一种原代小胶质细胞培养与纯化方法的改良

A modified method of microglia culturation and purification

目的建立一种简易高效的新生大鼠小胶质细胞的原代培养和纯化方法,提高小胶质细胞的产量以及纯度。方法在传统Miriam Mecha的混合胶质细胞培养方法的基础上通过改良操作,混合培养小胶质细胞,并分别采用手拍法、摇床法以及温和胰酶消化法,纯化小胶质细胞,通过差速贴壁进一步纯化细胞。荧光显微镜下标记小胶质细胞的特异性标记物Iba1、CD11b/c进行鉴定。结果 (1)分离培养第1天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;(2)纯化后小胶质细胞多呈静息状态,细胞免疫荧光CD11b/c、Iba1双阳性;(3)不同纯化方法细胞免疫荧光并计数显示:手拍法纯化小胶质细胞,细胞阳性率(>96%)明显高于胰酶消化法(>90%,P<0.05),前者细胞产量明显高于摇床法(P<0.05)。结论通过改良传统Miriam Mecha的混合小胶质细胞的培养和纯化方法,可以提高小胶质细胞产量以及纯度。

Objective To establish a simple and efficient method of primary microglial cell culture and purification of neonatal rats,improve the production and purity of microglial cell. Methods Improved operation,mixed cultured microglial cell and purified microglial cell respectively,on the basis of traditional hybrid Miriam Mecha microglial cell cultural method,by mild digestion with trypsin,shaking table method and hand clapping. Meanwhile,we also used differential attachment method to further purify cells. Purified microglia cells were labeled with specific marker： Ibal,CD11 b/c by fluorescence microscope. Results（ 1） Isolated purified primary microglia cell appeared irregular rounds and refractive uneven in1 day,then the cells gradually grown unipolar or multipolar process in 3 ~ 5 days,interestingly,some cells changed to stationnary state and became cladodromous in 7 days;（ 2） Purified microglia cells were labeled with specific marker： Ibal,CD11 b/c by fluorescence microscope and most of them changed to stationnary state and became cladodromous;（ 3） The three methods of identifying cells were detected by immunofluorescence,the result shown： The hand clapping method had a higher positive rate（ 95%） than trypsinization method and shaking table method（ 90%,P 0. 05） in different time points. Conclusion We improved the production and purity of primary microglia cells through this improved traditional Miriam Mecha method. It can get a greater number and purity of microglia cell,using hand clapping method combine with differential attachment method to purify mixed cultured cells.