大鼠脊髓少突胶质前体细胞的培养与鉴定方法

目的 探讨大鼠脊髓少突胶质前体细胞（OPCs）的分离纯化及诱导分化方法.方法 取出生后3d内SD大鼠脊髓,采用0.125％胰蛋白酶消化获取原代混合胶质细胞,培养10 d左右在37℃恒温摇床采取180 r/min摇速振摇并差速贴壁40 min获得纯化的OPCs;取纯化后培养3d的OPCs免疫荧光鉴定细胞纯度或诱导分化.结果 采用胰蛋白酶消化法获取原代混合胶质细胞、振摇并差速贴壁法分离纯化能够得到纯度较高的OPCs,折光性强,呈双极或三极形态,免疫荧光染色显示95％细胞表达A2B5和NG2（OPCs标志物）,经诱导分化后表达O4及MBP（少突胶质细胞标志物）.结论 采用振摇差速贴壁法可从大鼠脊髓中分离纯化获得高纯度的OPCs,获得的OPCs体外生长稳定,经诱导可分化为成熟的少突胶质细胞.

Isolation and identification of oligodendrocyte precursor cells of rat spinal cord

Objective To explore the improvement method of isolation and identification of oligodendro cyte precursor cells of neonatal rat spinal cord in vitro.Methods The mixed glial cells from SD rats spinal cord were obtained by using the 0.125％ trypsin digestion.About 10 days later,OPCs were isolated and puri fied by the shaking process and differential adhesion,and cultured in OPCs medium.The growth pattern of OPCs in vitro was observed by inverted microscope,and immunofluorescence assay was applied to identify the OPCs with NG2,A2B5 and the oligodendrocytes with O4 and MBP.Results Purfied OPCs were obtained in this method by the shaking process and differential adhesion.Enriched OPCs are phase bright with typical bi polar or tripolar morphology.More than 95％ of the cells were NG2 and A2B5 positive.Meanwhile,the OPCs can be induced differentiation to oligodendrocytes,which were O4 and MBP positive.Conclusions OPCs can be separated and purified by shaking and differential adhesion from SD rats spinal cord.Meanwhile,OPCs can be induced differentiation to oligodendrocytes in vitro.