小胶质细胞和少突胶质细胞前体的培养和鉴定

目的 探讨新生大鼠脑组织小胶质细胞(MG)和少突胶质细胞(OL)前体的分离和体外培养方法 . 方法 取新生2 d SD大鼠脑组织,体外原代培养混合胶质细胞7 d后,分别采用"改良振荡伴差速贴壁"法和"营养缺失伴振荡"法纯化培养MG和OL前体,并分别应用免疫荧光染色异凝集素-B4(IB4)和OL前体标记物(O4)进行鉴定.结果 混合胶质细胞培养7 d后呈明显三层增长,其中MG位于上层,星型胶质细胞位于底层,两者之间为2型少突星型(O2A)祖细胞.纯化培养后OL前体胞体呈小圆形,有双极或三极突起,MG则以阿米巴形、圆形居多,或边缘呈毛刺状.免疫荧光染色IB4显示绿色荧光,MG纯度达到90%以上.免疫荧光染色O4显示棕黄色荧光,OL前体纯度达到95%以上. 结论 采用"改良振荡伴差速贴壁"法以及"营养缺失伴振荡"法分别成功获取大量纯度高、活力好的MG和OL前体.

Culture and identification of microglias and preoligodendrocytes

Objective To explore the isolated and in vitro cultural methods of preoligodendrocytes (preOLs) and microglias (MGs) obtained from the brain tissues of neonatal rats.Methods The MGs and preOLs isolated from the brain tissues of the 2-d-old SD neonatal rats were primarily cultured by using the nutrition-deficient method with the combination of shaking and the modified shaking and adherence method, respectively. The purity of the cultured cells was identified by immunocytochemical analysis. Results After cultured for 7 d, the mixed glias formed 3 cell layers consisting of the microglias in the upper layer, O2A progenitor cells in the middle layer and the astrocytes in the basal layer, respectively. It was observed under fluorescence microscope that the cultured preOLs were small and round with bi-polar or tri-polar prominence, and the cultured microglias displayed amebiform or round morphologies, sometimes with the burr rim shape. The immunocytochemical analysis identified that the purities of the cultures were consistently ＞95% for O4 positive labeled preOLs and ＞90% for FITC-labeled IB4 positive MG. Conclusion By using the nutrition-deficient method with the combination of shaking and the modified shaking and adherence method, the massive highly-pure and alive microglia and preOLs are obtained successfully.