大鼠脊髓小胶质细胞体外纯化培养方法的建立

Primary Culture and Purification of Microglia from Rat Spinal Cord

目的：建立一种简易高效的大鼠脊髓小胶质细胞原代培养方法。方法：取新生SD大鼠的脊髓进行脊髓胶质细胞的原代混合培养，第3天换液一次，此后一直不换液。至第10天左右脊髓小胶质细胞长满后，在恒温摇床上振摇（37℃、180 rpm、1~2 h），并采用差速粘附20~40 min，纯化脊髓小胶质细胞。用Iba1和CD11b的免疫荧光染色对分离纯化24 h后的小胶质细胞进行纯度鉴定。结果：成功分离纯化大鼠脊髓小胶质细胞。细胞体呈圆形或者梭形，折光性很强。Iba1和CD11b免疫荧光鉴定结果显示小胶质细胞的纯度≥95%。结论：采取营养剥夺，以及振摇和差速粘附法建立了一种高产量、高纯度的脊髓小胶质细胞培养方法。

Objective： To establish a simple and highly efficient primary culture method for microglia from rat spinal cord. Methods： Mixed glial cells were obtained from the spinal cord of newborn SD rats. On day 3, the culture medium was changed until the cultured microgila became fused on day 10. The microglia were purified using shocking methods （37 ℃, 180 rpm, 1~2 h） with different adhesion time （20~40 min）. The isolated and purified microglia were identified with Iba1 and CD11b after 24 h. Results： The rat spinal cord microglia were successfully isolated and purified. The cultured microglia presented round or fusiform, highly refractive morpholo-gy. Iba1 and CD11b fluorescence tests showed that the purity of microglia was more than 95%. Conclusion：Using nutrition deprivation, Shock and different adhesion time methods, a primary spinal cord microglia culture method with high yield and purity was established, providing a useful tool for studying rat spinal cord microglia in vitro.