原代培养星形胶质细胞和小胶质细胞的特性与鉴定

Characterization and identification of primarily cultured astrocytes and microglia

摘要:

本研究旨在明确原代培养的星形胶质细胞和小胶质细胞不同代次的生长特性，优化高效获取状态一致细胞的技术方法。将新生乳鼠的脑组织进行原代分离培养胶质细胞，通过细胞增殖检测试剂盒（cell counting kit-8，CCK-8）测定混合胶质细胞增殖曲线，使用流式细胞术检测两类细胞比例，并通过免疫荧光染色鉴定两类胶质细胞分型情况。生长曲线显示P0和P1代混合胶质细胞增殖活力最好；通过170 r/min机械振摇30 min可获得97.3%的高纯度小胶质细胞，该纯化方法得到的P0、P1、P2代离子钙接头蛋白-1（ionized calcium-binding adapter molecule 1，Iba-1）阳性小胶质细胞的形态及其M1、M2表型比例无代次差别；通过星形胶质细胞表面抗原-2（astrocyte cell surface antigen-2，ACSA-2）磁珠抗体分选的方法可获得纯度达到95.7%的星形胶质细胞，该纯化方法得到的P0、P1、P2代胶质纤维酸性蛋白（glial fibrillary acidic protein，GFAP）阳性星形胶质细胞的形态及其A1、A2表型比例无代次差别。本研究详述了原代分离培养的小胶质细胞和星形胶质细胞的生长特点，证明了获取两类胶质细胞的最佳代次，优化了获取两类胶质细胞的技术方法，验证了连续培养两代不会影响其功能表型。本结果为研究神经系统炎症相关疾病的分子机制提供了技术支撑。

Abstract:

The aim of this study was to investigate the growth characteristics of primarily cultured astrocytes and microglia of different generations and then optimize the method for obtaining primary astrocytes and microglia effectively. Primarily cultured microglia were isolated and purified from the cortices of neonatal mice. The proliferation curve of mixed glia cells was measured by Cell Counting Kit-8 (CCK-8) assay, the proportion of astrocytes and microglia was detected by flow cytometry, and the polarization of the two types of glia cells was identified by immunofluorescence staining. Cell growth results showed that the mixed glia cells of P0 and P1 generation had the best proliferative activity; 97.3% of the high purity microglia could be obtained by mechanical shaking at 170 r/min for 30 min, and there was no significant difference in the morphology of ionized calcium-binding adapter molecule 1 (Iba-1) positive microglia and the proportion of M1 and M2 phenotype among the P0, P1 and P2 generations of microglia isolated by the above methods. Moreover, 95.7% of the high purity astrocytes could be obtained by astrocyte cell surface antigen-2 (ACSA-2) magnetic beads separation, and there was no significant difference in the morphology of glial fibrillary acidic protein (GFAP) positive astrocyte and the proportion of A1 and A2 phenotype among the P0, P1 and P2 generations of astrocyte isolated by the above methods. Taken together, this study observed the growth characteristics of primarily cultured microglia and astrocyte in vitro, and then proved the best generations for purifying microglia and astrocytes. Finally, we optimized the methods of obtaining microglia and astrocyte, and verified that continuous culture within 2 generations will not affect the functional phenotypes of glia cells. These results provide technical support for studying the molecular mechanism of inflammation-associated diseases in nervous system.