新生大鼠少突胶质前体细胞低糖缺氧损伤模型的建立

The establishment of oligodendrocyte precursor cells low glucose and oxygen deprivation model in neonatal rat

目的:利用连二亚硫酸钠(Na2S2O4)建立少突胶质前体细胞(oligodendrocyte precursor cells,OPCs)低糖缺氧损伤模型。方法:取新生1 d Sprague-Dawley(SD)大鼠大脑皮质,体外混合及纯化培养获得OPCs,O4免疫荧光染色鉴定;取纯化2 d的OPCs,分为正常组和Na2S2O4损伤组,Na2S2O4损伤组又分为0.5、1 h和1.5 h三组,倒置显微镜下观察各组细胞形态;CCK-8法检测细胞存活率;透射电镜观察细胞超微结构。结果:免疫荧光染色鉴定显示纯化的细胞绝大部分为O4阳性;形态学观察显示,缺氧0.5 h,大部分细胞突起回缩,胞体变圆,颜色变暗,随低糖缺氧损伤时间的延长OPCs逐渐脱壁,1.5 h时只有少数贴壁细胞;CCK-8法检测显示损伤0.5、1 h和1.5 h组细胞的存活率显著降低,与正常组比较均有统计学意义,损伤后1.5 h与0.5 h比较,细胞的存活率具有显著性差异;电镜观察损伤1.5 h后的细胞,线粒体肿胀,有些细胞胞核固缩成块,细胞器破碎。结论:用浓度为2mmol/L Na2S2O4的低糖培养基处理OPCs 1.5 h可以建立有效的低糖缺氧损伤模型。

Objective：This study was design to establish oligodendrocyte precursor cells（OPCs） low glucose and oxygen deprivation model with sodium dithionite（Na2S2O4） in neonatal rat.Methods：The OPCs from cerebral cortex of newborn 1 day Sprague-Dawley（SD） rat were cultured and purfied and identified by immmunofluorescent staining with O4 antibody.The purified OPCs were divided into normal group and model group after being cultured 2 days,and the model group was further divided into 0.5,1 and 1.5 h groups.The morphology of OPCs was observed by using inverted microscope and the viability of OPCs was measured by Cell Counting Kit-8（CCK-8） assay.The ultrastructure of OPCs was analyzed by transmission electron microscope.Results：Immunofluorescence staining showed the purified cells almost entirely were O4-positive.The processes of OPCs began to shrink and cell bodies became into round with Na2S2O4 treated for 0.5 h.With the extension of culture time,OPCs floated and only few cells still attached to the flask after 1.5 h Na2S2O4 treatment.The survival rate of OPCs significantly decreased in the model group compared with that of the normal group.And there was also statistically diflerence between 1.5 h group and 0.5 h group.The mitochondria of the OPCs swelled and the cell nucleus condensated and some organelles breaked into pieces under electron microscope.Conclusion：The OPCs low glucose and oxygen deprivation model in neonatal rat could be established with 2 mmol/L Na2S2O4 treated for 1.5 h.