一种连续获取大量高纯度小胶质细胞的培养方法

A novel method for successive acquisition of high purity and yield of microglia

目的建立一种简易、高产量高纯度的连续获得小胶质细胞的培养方法。方法原代培养新生1～3dWistar大鼠大脑皮层中混合胶质细胞，第8-9天采用振摇法和玻璃吸管吹打法分离获得Yield1小胶质细胞，按1：2传代培养剩余贴壁的混合胶质细胞，分别继续培养10～12d、12～14d后应用同样方法获得Yield2、Yield3小胶质细胞：采用流式细胞仪分析所获小胶质细胞CD11b／c、CD45、CD80、CD86、GFAP的表达，免疫荧光染色和CCK-8法分别检测小胶质细胞CD11b／c的表达和增殖情况，应用扫描电镜观察其超微结构，应用吞噬红色乳胶微球实验评价其吞噬功能。结果本实验所用培养方法连续稳定地获得了大量高纯度的小胶质细胞：流式细胞仪检测结果显示Yield1与Yield3小胶质细胞CD11b／c、CD45、CD80、CD86表达阳性：免疫荧光染色显示Yield1、Yield2、Yield3小胶质细胞CD11b／c表达均阳性；CCK-8法结果显示3代小胶质细胞增殖活力相比较差异无统计学意义（P〉0．05）；扫描电镜显示3代小胶质细胞形态均呈胞体饱满、胞突细长等细胞形貌特点。相同条件下Yield1、Yield3小胶质细胞之间吞噬功能比较差异无统计学意义（P〉0．05）。结论以本实验方法可以连续获得大量高纯度的小胶质细胞，且这些不同代次小胶质细胞的抗原表型、增殖活力及吞噬功能无明显差异，可为相关的实验研究提供细胞来源。

Objective To establish an easy culture method of successively getting high purity and yield ofmicroglia. Methods Cortices of neonatal Wistar rats （1-3 days old） were employed in this experiment. The first-generation microglial cells were isolated from the mixed glial culture by mechanical means （gently shaking and blowing with pipette）. After the mixed glial cells being passaged at a density of 1：2 at 2 successive times （10-12 d and 12-14 d of continuous cultures）, respectively, the second and third generations ofmicroglial cells were harvested. CD11b/c, CD45, CD80, CD86 and GFAP were employed as the identification markers in detecting the phenotypes and purity of different generation of microglial cells by scanning electron microscope and flow cytometry. Immunofluorescence staining and CCk8 vitality measurement were used to judge the expression of CD11b/c and detect the proliferation of microglia cells. Microglial phagocytotic function was evaluated by phagocytosis of fluorescent microspheres. Results High yield and purity of microglial cells were stably obtained in this experiment. CD1 lb/c, CD45, CD80 and CD86 positive expressions were noted in the first and thirdgenerations of microglial cells by flow cytometry; CD11b/c positive expression was noted in the first, second and third generations of microglial cells by immunofluorescence staining. No obvious differences in the 3 different generations of microglia cells were found on proliferation ability by CCk8 vitality measurement, and on morphology and phenotypes by scanning electron microscope; no obvious differences in the first and third generations of microglia cells were found on phagocytic ability （P〉0.05）. Conclusion High yield and purity ofmicroglial cells can successively obtain through the above method; no significant differences are noted among different generations ofmicroglia cells on purity, morphology, phenotypes, proliferation activity and phagocytic ability,