吴雪,黄彩云,袁佳钰,付雅芬,马佳雪,杨洁,张前
WU Xue,HUANG Caiyun,YUAN Jiayu,FU Yafen,MA Jiaxue,YANG Jie,ZHANG Qian

• 1:陕西中医药大学药学院
• 2:陕西师范大学生命科学学院

摘要(Abstract)：

目的 建立一种简便、高效可同时得到小鼠脊髓源的原代星形胶质细胞与小胶质细胞的分离、纯化、培养方法。方法 显微镜下剥离胎鼠脊髓，剪碎后经机械吹打、自然沉降制成细胞悬液，接种于多聚赖氨酸包被的培养瓶，用含10%胎牛血清的DMEM高糖培养基培养得到混合胶质细胞；当细胞汇合度> 90%时，采用恒温定轨摇床振荡结合传代除杂的方法获得星形胶质细胞与小胶质细胞；采用光学显微镜观察纯化前后的细胞形态，采用免疫荧光法检测细胞标志物GFAP（星形胶质细胞）、CD11b（小胶质细胞）并鉴定其纯度。结果 本方法通过一次操作，同时获得了2种脊髓源的胶质细胞，得到的星形胶质细胞形态正常，活力好，可在1～2 d铺满培养瓶底，细胞阳性率> 97%；得到的小胶质细胞呈多分枝状，细胞间相互交织成网状，细胞阳性率> 97%；采用免疫荧光法鉴定P1星形胶质细胞和P0小胶质细胞均可见绿色荧光的阳性细胞、细胞核为蓝色荧光。结论 采用可在较恒温定轨摇床振荡结合传代除杂的方法短时间（6～9 d）内同时获得2种高纯度的胶质细胞。
Objective To establish a simple and efficient method to isolate, purify and culture primary astrocytes and microglia from fetal mouse spinal cords.Methods The spinal cords were dissected from fetal mice under a microscope. The spinal cords were minced, disassociated by pipetting blow and naturally precipitated to obtain dispersed cell suspension. Cell suspension was seeded in a T25 cell culture flask coated with poly-L-lysine, and cultured with high-glucose DMEM supplemented with 10% fetal bovine serum to obtain mixed glial cells. When the confluence of these cells was over90%, the T25 cell culture flask was placed on a orbital shaker for contionous cell culture and mixed glial cells were passaged for purification. The resulting cells were astrocytes and microglia. Optical microscope was used to observe the morphological changes of cells before and after purification. Cells were immunofluorescently stained with anti-GFAP( astrocyte marker) or anti-CD11b( microglia marker) to identify astrocytes and microglia, and analyze their purity.Results Two types of glial cells were simultaneously obtained through one experiment. Cell morphology of the astrocytes was normal. The astrocytes had good cell viability and grew to the confluence within 1-2 days, and the positive rate was over 97%. The resulting microglia exhibited highly ramified morphology, and intertwined with each other to form a reticulate structure, and the positive rate was over 97%.Immunofluorescent staining showed that green fluorescent positive cells and blue fluorescent nuclei were observed in P1 astrocytes and P0 microglia.Conclusion Orbital shaking combined with cell passaging under constant temperature can be used to remove non-glial cells and simultaneously obtain two types of glial cells with high purity in a short time(6-9 d).

关键词(KeyWords)： 脊髓;胎鼠;星形胶质细胞;小胶质细胞;原代培养
spinal cord;fetal mice;astrocytes;microglia;primary culture

Abstract:

Keywords:

基金项目(Foundation): 陕西师范大学中央高校基本科研业务费专项资金项目（GK202103079）;; 国家大学生创新计划项目（S202110718062）

作者(Author): 吴雪,黄彩云,袁佳钰,付雅芬,马佳雪,杨洁,张前
WU Xue,HUANG Caiyun,YUAN Jiayu,FU Yafen,MA Jiaxue,YANG Jie,ZHANG Qian

DOI: 10.19367/j.cnki.2096-8388.2022.04.010

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