人小胶质细胞的分离、纯化、特异分子表达与吞噬功能研究

Studies on the isolation,purification,specific molecule expression and phagocytosis of human microglia

目的:探讨人小胶质细胞(microglia)分离、纯化、培养及鉴定的方法,为深入研究小胶质细胞功能建立一种简便的、稳定的细胞模型。方法:取5个月人工药物引产的胎儿脑组织,将脑组织剪碎、研磨、过滤、离心获取混合胶质细胞悬液,用DMEM/F12完全培养基调整细胞密度为2×1010cells/L,接种于75cm2培养瓶中(记作first0d);7-10d后,进行第1次纯化。此次纯化采用轻柔摇动法将贴壁不牢的小胶质细胞和少突胶质细胞与底层的星形胶质细胞分离并转入另一培养瓶继续培养扩增(记作second0d);4-5d后,混合的小胶质细胞和少突胶质细胞汇合成片,采用胰酶-EDTA消化法结合差速贴壁法进行第2次纯化。此次纯化将消化得到的细胞悬液离心和重悬,接种于培养瓶(记作0h);2h后,小胶质细胞贴壁生长,少突胶质细胞依然漂浮在培养上清中,此时通过移走上清以去除少突胶质细胞,即得到高纯度的小胶质细胞;运用激光共聚焦显微镜技术、流式细胞术结合细胞表面或胞内免疫荧光抗体染色技术检测所分离的小胶质细胞CD45,CD11b和CD68的阳性率,以计算其纯度;通过吞噬荧光微球评价其吞噬功能;利用胞膜边缘波动(membrane ruffling)现象结合荧光标记的鬼笔环肽染色技术鉴定其活化水平。结果:我们所分离的小胶质细胞中,>98%的细胞表达CD45,CD11b和CD68,并且能够吞噬荧光微球。结论:我们成功地分离和纯化了人小胶质细胞,所得到的小胶质细胞高表达CD45,CD11b和CD68分子,并且具有很强的吞噬功能,为进一步进行其功能和蛋白质组研究奠定基础。

AIM ： To explore a method of isolation, purification, culture and identification of human microglia.METHODS： The brain tissue from abortive fetus was sterilely obtained, then chopped and triturated gently. The suspension was filtered and centrifuged to separate and isolate mixed glia cells. The cell density was adjusted to 2×10^10cells/L with DMEM/F12 complete medium and cultured in 75 cm^2 flask at 37℃ in CO2 incubator （marked as first 0 d）. After 7 -10 days culture, floating cells including microglia and oligodendrocytes appeared in the flask. The first time purification was carried out to obtain highly purified microglia, for which the flasks were shaken gently. The floating cells were collected and transferred into a new flask （ marked as second 0 d）. 4 - 5 days later, when microglia and oligodendrocytes grew in confluent state, the second purification was performed, for which the mixed cultured cells were digested with trypsin - ED- TA and cell suspension was centrifuged, then the cell pellet was suspended by DMEM/F12 complete medium and cultured in flask （ marked as 0 h）. After 2 h, non - microglial cells including oligodendrocytes and cell debris were removed, and fresh medium was added into the adherent cells. In this way. the whole procedure of microglia purification was completed and the highly purified microglial cells were obtained. After purification, the expression rate of CD45, CD11 b and CD68 on/in microglia were detected by laser - confocal microscopy and flow cytometry on the basis of immuno - fluorescence stai- ning to identify the purity of microglial cells. Microglial phagocytotic function was evaluated by phagocytosis of fluorescent microspheres. The level of activation was identified by the phenomena of membrane ruffling in combination with fluorescent phalloidin staining. RESULTS： More than 98% of the microglias that we isolated and purified expressed CIMS, CD11 b and CD68. Almost all of the microglias could phagocytize fluorescent microbeads CONCLUSION： We succeed in isolating and purifying human microglias,which express CD45 ,CD11 b and CD68. Almost all of the cells exhibit phagocytic function. So these cells will be useful for further functional and proteomic studies.