构建一种高产量小胶质细胞体外纯化培养的方法

背景小胶质细胞在一些神经系统疾病如帕金森病、阿尔茨海默病中有重要作用.体外原代培养是进行小胶质细胞功能研究的基础,但目前常用的一些经典培养方法存在步骤繁琐、产量过低的问题.目的建立一种简易高产量的原代小胶质细胞体外纯化培养方法.设计以细胞为单一样本的探索性研究.单位华中科技大学同济医学院附属协和医院神经内科.材料实验于2004-04/10在协和医院中心实验室完成,新生(出生1d)昆明小鼠10只,雄性.方法改进的培养方法以McCarthy等的经典培养方法为基础,通过提高初次接种密度,减少细胞离心过滤过程,进行不换液的营养缺失培养等重要改进,并使用低浓度胰酶-乙二胺四乙酸消化法分离纯化小胶质细胞,经MAC-1抗体免疫化学染色标记,计算MAC-1抗原阳性细胞,观察小胶质细胞培养的产量和纯度.主要观察指标①倒置显微镜观察小胶质细胞形态.②免疫组织化学鉴定比较两种方法下小胶质细胞纯度、激活状态.结果经典的McCarthy培养方法小胶质细胞培养周期为20d,产量为2×105个/瓶,细胞纯度为95%～97%;改进的方法小胶质细胞培养周期为15d,产量为1×106个/瓶,得到的小胶质细胞纯度为96%～98%.与经典的McCarthy培养方法比较,改进的方法周期短,产量提高了3～10倍.两种方法培养的小胶质细胞纯度和细胞激活状态没有明显差别.结论新方法得到的小胶质细胞纯度与激活状态均与经典方法相似,但由于其步骤简单,周期短和有效提高了培养产量,为进行小胶质细胞功能特性以及在神经修复中作用的深入研究提供了良好的方法学基础.

A new method forculturing highly enriched microglia in vitro

BACKGROUND: Microglial cells are prominently involved in certain neurologic diseases such as Parkinson disease and Alzeheimer disease. In vitro primary culture is commonly used in studies on the functions of microglia.However, these classical culture methods have some defects including complex procedures and low out-put.OBJECTIVE: To establish a simplified high-output primary culture of microglia.DESIGN: An explorative experiment with microglial cells as the single sample.SETTING: Department of Neurology, Affiliated Union Hospital, Tongji Medical College, Huazhong University of Science and Technology.MATERIALS: The study was finished at the Central Laboratory of Union Hospital from April to October 2004. Microglial cells were obtained from 10 newborn(one day) male Kunming mice that were selected.METHODS: The author' s culture method was based on McCarthy method, we developed a new culture method and made some improvements,including the increased cell density for primary culture and nutritional deprivation. The microlglial cells were isolated with low-concentration trypsin-EDTA(ethylene diamine tetraacetic acid) digestion and immunochemically labeled with MAC-1 antibody, so as to measure the output and purity of microglia.MAIN OUTCOME MEASURES: ① Morphologic features of microglial cells, observed with inverted microscope; ② Purity and activity of microglia cultured with these two methods, were measured immunohistochemically.RESULTS: For microglia cultured with McCarthy method, the culture cycle was 20 days and the output was 2 × l05 cells per flask with a purity of 95% -97%. The new method shortened the culture cycle to 15 days and the output reached 1 × 106 cells per flask with a purity of 96-98%. Cell purity and activity had no significant difference between these two culture methods.CONCLUSION: The new method has a similar purity and activity with classical method; however, it may simplify procedures, shorten cycle, and increase output, and therefore can be a useful method for studies on microglia function and for nerve repair.