树鼩大脑皮层少突胶质前体细胞的分离鉴定

Isolation and Identification of Oligodendrocyte Precursor Cells from Tree Shrews Cerebral Cortex

目的建立树鼩大脑皮层少突胶质前体细胞(OPCs)的体外分离、原代培养方法,以及鉴定技术。方法新生48 h内给树鼩施行安死术后采集大脑,去除脑膜和脑干,留下大脑皮层,然后使用胰酶和DNase I联合消化大脑皮层,分离细胞原代培养。通过采用化学分离法和差速贴壁法从原代培养中获得较为纯化的OPCs。对纯化和定向分化的细胞分别进行硫酸软骨素蛋白多糖4(NG2)、髓鞘碱性蛋白(MBP)细胞免疫荧光染色鉴定。结果纯化后的OPCs,胞体圆形或椭圆形,折光性强,有两极或三极突起,经NG2细胞免疫荧光染色,细胞纯度达97%左右。加入分化培养液后,OPCs可分化发育成熟,经MBP细胞免疫荧光染色为阳性。结论成功建立了树鼩大脑皮层OPCs的体外分离纯化培养方法。

Objective To establish an effective method of isolation, identification and primary culture for oligodendrocyte precursor cells(OPCs) which derived from the cerebral cortex of tree shrews in vitro. Methods Newborn tree shrews within 48 hours were given euthanasia, the cerebral cortex were collected, removed the meninges and brainstem. Then, the cerebral cortex was digested with trypsin and DNase I, and primary cultured. Oligodendrocyte precursor cells were purified by chemical separation and differential adherence. The oligodendrocyte precursor cells were identified by immunofluorescence staining of chondroitin sulfate proteoglycan 4(NG2) and myelin basic protein(MBP). Results The purified oligodendrocyte precursor cells showed round or oval cell body, with strong refractive and bipolar or tripolar protuberances. The purity of the cells was 97% by immunofluorescence staining of NG2. After adding differentiation medium, oligodendrocyte precursor cells could differentiate and mature, and the immunofluorescence staining of MBP is positive.Conclusion A method for isolation and purification of cerebral cortex oligodendrocyte precursor cells from tree shrews was successfully established.