少突胶质前体细胞的培养、鉴定及巨噬细胞移动抑制因子对其促增殖作用研究

Isolation and identification of neonatal rat oligodendrocyte precursor cells and proliferation by macrophage migration inhibitory factor

目的少突胶质细胞是中枢神经系统的重要组成部分。本文探讨获取小鼠高纯度少突胶质前体细胞系的培养及鉴定方法,以及巨噬细胞移动抑制因子(MIF)对其增殖活性的调节。方法取新生(0-2 d)C57B6小鼠的大脑皮层进行混合胶质细胞培养,在9-10 d时采用振荡、差速贴壁的方法分离、纯化少突胶质前体细胞,然后用添加了神经营养因子(N2)、血小板衍生生长因子-AA(PDGF-AA)、碱性成纤维细胞生长因子(bFGF)的无血清培养基培养。倒置显微镜下每天观察细胞的生长情况,免疫荧光法对表面抗原进行细胞鉴定。然后按104个细胞/孔加入96孔板培养,细胞70%-80%融合时用不同浓度MIF处理细胞,48 h后用甲基噻唑基四唑(MTT)检测细胞增殖程度。结果获得纯度90%以上少突胶质前体细胞(OPCs),少突胶质前体细胞NG2及血小板衍生生长因子受体(PDGFR)抗体阳性;胶质细丝酸性蛋白(GFAP)及髓鞘碱性蛋白(MBP)均为阴性。MTT检测结果显示低剂量的MIF能促进OPCs的增殖,并具有剂量效应关系;高剂量MIF则抑制OPCs的增殖。结论通过振荡及差速贴壁法并结合少突胶质细胞定向培养基可以成功获得高纯度的OPCs,MIF能以剂量效应关系促进OPCs增殖。为进一步深入探讨其相关机制及OPCs移植治疗脊髓损伤奠定基础。

Objective To explore the method of isolation and identification of neonatal mouse pups oligodendrocyte precursor cells and research on its proliferation by macrophage migration inhibitory factor（MIF）.Methods The mixed glial cells from the cerebral cortices of C57B6 neonatal mouse pups were cultured.Until 9-10 days OPCs were isolated and purified by the shaking process and differential adhesion,and then cultured in defined serum-free medium,with appended neurotrophin 2（N2）,platelet-derived growth factor-AA（PDGF-AA）,basic fibroblast growth factor（bFGF）.The growth pattern of OPCs in vitro was observed by inverted microscope,and immunofluorescence assay was applied to identify the OPCs with NG2,myelin basic protein（MBP） and glial fibrillaryacidic protein（GFAP） antibodies.Finally,the proliferation of the pure OPCs populations were detected by MTT.Results The distinct stratification of OPCs and astrocytes developed around 9-10 days in primary culture.90% of cultured OPCs were obtained.The OPCs were NG2 and PDGFR positive,while GFAP and MBP were negative.Low dose MIF could promote OPCs proliferation significantly in a dose-dependent manner,but high dose MIF inhibits OPCs proliferation.Conclusion OPCs can be separated and purified by shaking and differential adhesion and OPCs can be maintained as immature precursor cells in defined serum-free medium.Meanwhile,MIF can stimulate OPCs proliferation in a dose-dependent manner suggests that MIF play an important role in the central nervous system development and injury repair.