大鼠皮层神经元原代培养及毒胡萝卜素干预后的表现

Primary culture of isolated rat cerebral cortex neuron and characteristics of neuron after interference with thapsigargin

目的探讨离体大鼠皮层神经元原代培养方法及毒胡萝卜素诱导大鼠皮层神经元内质网应激的作用特点。方法体外培养SD乳鼠皮层神经元细胞,免疫组织化学、免疫荧光染色鉴定神经元纯度,Hoechst33258单染法观察凋亡细胞核的形态变化及测出细胞凋亡率,透射电子显微镜观察神经元的亚细胞结构。结果 SD乳鼠皮层神经元可纯化体外培养,对培养7d后的神经元进行NSE免疫荧光染色显示计数NSE阳性细胞数占细胞总数的百分数为(95±3)%,在TG浓度为2μmol/L作用时间为24h时离体培养原代神经元细胞凋亡率最高为19%±0.54%,P<0.05。神经元细胞在2μmol/LTG作用24h后神经元呈现典型凋亡形态学改变,核周内质网,线粒体结构模糊不清,包膜破损,且出现染色质浓集、核固缩、细胞皱缩、核碎裂。结论体外改良的培养方法可以获得较高纯度的神经元,在毒胡萝卜素诱导的神经元内质网应激时,线粒体改变引起的变化可能是下游事件。

Aim To explore the primary culture of isolated rat cerebral cortex neuron and the stress characteristics of cortical neurons endoplasmic reticulum induced by thapsigargin.Methods The cortical neurons of SD suckling mouse cultured in vitro,were indentified by immunohistochemical and immunofluorescence stain,the morphological change of apoptotic nucleus and measure cell apoptosis rate,the morphological change of apoptotic nucleus and measure cell apoptosis rate,were observed by Hoechst 33 258 simple staining and the the neurons subcellular structure was observed under a transmission electron microscope.Results The cortical neurons of SD suckling mcie was purified and cultured in vitro,the neurons were stained with NSE immunofluorescence stain and the total NSE positive cells 7 days after culture accounted for（95 ± 3）%.The apoptosis rate of in vitro primary culture neurons,in the 2μmol TG for duration of 24h was the highest of 19%± 0.54（P 0.05） with typical apoptosis morphological changes in 2μmol/lTG for a duration of 24h in addition to the nebulous structure of perikaryon endoplasmic reticulum,mitochondria and the membane breakage,enriched chromatin,karyopyknosis,cells shrink,nucleus splintered as well.Conclusion The modified culture in vitro allow to obtain higher purified neurons.The morphological changes of mitochondria may be due to downstream events at stress of cortical neurons endoplasmic reticulum induced by thapsigargin.