乳鼠脑小胶质细胞原代培养与鉴定

Primary Culture and Identification of Microglias from Neonatal Rat Brain

目的:探索乳鼠小胶质细胞的原代培养和鉴定方法。方法:取新生SD乳鼠脑组织捣碎、消化及共培养,用振摇法分离纯化小胶质细胞并继续培养;在相差显微镜下观察共培养第3、9及14天时细胞形态,计数纯化培养后小胶质细胞数及存活率,采用免疫细胞化学染色方法在共聚焦显微镜下计数纯化培养后小胶质细胞特异性OX-42抗体表达阳性的细胞数及存活率,同时观察小胶质细胞活化形态;观察纯化培养12、24、48及72 h后小胶质细胞的静止型细胞数和静止率。结果:振摇法共培养第14天时可见胞体折光不均的星形胶质细胞最多,分离时可获得1.2×106个/瓶(75 cm2,250 m L)小胶质细胞,免疫细胞化学染色显示小胶质细胞特异性OX-42表达阳性细胞达95%,存活率>95%;在显微镜下小胶质细胞形态以阿米巴样、长梭形及马鞍形为主;纯化培养72 h后小胶质细胞的静止型细胞数和静止率最高。结论:振摇法分离小胶质细胞纯化培养72 h,可获得最高的静止型小胶质细胞数和静止率。

Objective： To explore the method of primary culture and identification of microglia cells from the neonatal rats. Methods： The brain tissues from the neonatal SD rats were stamped,digested and co-cultured. The microglia cells were isolated and purified by shaking method and continued to be cultured. The cellular morphology was observed under phase contrast microscope at day three,day nine and day fourteen after co-culture,and the number of microglia cells was counted and survive rate was detected after purification and culture. Immunocytochemical staining method was adopted to count cell number of positive expression of specific OX-42 antibody of microglia cells and survive rate after purification and culture under confocal microscopy. The morphology was observed by the inverted phase contrast microscope,and the resting cell number and resting rate 12,24,48 and 72 hours after purification and culture were counted. Results： It was observed that there were abundant astrocytes of uneven refraction 14 days after shaking method and co-culture. Immunocytochemical stain showed that the method steadily produced 1. 2 × 10^6 cells per flask（ 75 cm2,250 m L） with high survival rate（ 〉95%） and the high positive rate（ 〉95%） of expression of OX-42. Under the microscope,the morphology of microglia cells was mainly ameboid morphous,long spindle shape and saddle. The number of resting cells and the rate of resting cells were the highest 72 hours after purification and culture.Conclusion： The microglia cells were isolated and purified by shaking method and continued to be cultured for 72 hours and the highest resting cells number and resting rate can be obtained.