离体脊髓星形胶质细胞的纯化与鉴定.DOCVIP

临床医学论文-离体脊髓星形胶质细胞的纯化与鉴定 【摘要】? 目的 探索新生大鼠脊髓星形胶质细胞的分离、培养与传代及纯化方法，鉴定其培养纯度。方法 分离、培养新生大鼠脊髓星形胶质细胞，用换液时暴露结合旋转法加以纯化，酶消化法传代后进行免疫组化鉴定，并染核计数星形胶质细胞的纯度。结果 星形胶质细胞贴壁快，多在12 h内，3 d后数量显著增加；免疫细胞化学染色显示，NF200抗体染色为阴性，提示培养盖片中不含神经元；GFAP阳性染色细胞高达95.8%。结论 换液时短暂暴露结合恒温旋转法可纯化得到高纯度的星形胶质细胞，是一种简单、高效的星形胶质细胞纯化方法。 【关键词】? 星形胶质细胞；神经微丝蛋白；胶质纤维酸性蛋白 ??? Purification and identification of astrocyte of spinal cord in vitro ??? Abstract: Objective? To explore the method of isolation, culture, and purification of astrocyte derived from spinal cord of neonate rats, and identified by immunocytochemical stain. Methods?Astrocyte derived from spinal cord of neonate rats were isolated and cultured, purified by exposed a short time when exchanging medium and rotation. Passage through enzyme digestion and identified by immunocytochemical stain, to count the purity coefficient of astrocyte. Results?Astrocytes adhered after plated 12 h, the numbers increased significantly after 3 d. Immunocytochemical stain showed: there were no neuron on culture covers because the stain of NF200 was negative, while the positive rate of GFAP stain was up to 95.8%. Conclusion?High purified astrocytes could be got by exposed a short time when exchanging medium and rotation, so it is a simple and high effective method for the purification of astrocyte. ?牋 Keywords: astrocyte; neurofilament; glial fibrillary acidic protein ??? 近年，神经胶质细胞（astrocyte，AST）已成为神经科学研究的热点之一［1］，它不仅具有支持、分隔、营养和保护神经元的作用，而且参与了脑的高级功能活动，并在突触形成中起着重要作用［2-3］。AST的分离、培养方法报道较多，取材部位以大脑皮层为主，脊髓相对较少，纯化方法常以多次传代为主。因此，有必要对星形胶质细胞的分离、培养与传代和纯化进行系统的研究，并对其培养纯度进行鉴定。 ??? 1? 材料与方法 ??? 1.1? 试剂与仪器 ??? DMEM培养基、D-Hank液和胎牛血清（FBS）皆购自Gibco公司，L-谷氨酰胺、Hoechst、胰蛋白酶和小鼠抗神经微丝单克隆抗体（NF200抗体）购自Sigma公司，兔抗胶质纤维酸性蛋白（GFAP）、FITC标记山羊抗兔IgG、TRITC标记山羊抗小鼠-IgG和抗体稀释液皆购自北京中杉金桥生物技术有限公司。显微镜型号为Olympus BX-60，荧光激发装置为Olympus MODEL BH2-RFL-T3，CO2培养箱THERMO FORMA 3111。 ??? 1.2? 细胞的分离和培养 ??? 取新生24 h内Sprague-Dawleyr大鼠（购自第三军医大学大坪医院野战外科研究所实验动物中心），参照McCarthy等［4］的方法加以改进。取新生24 h内的SD大鼠酒精浴后在无菌条件下取脊髓，置冰浴D-Hank液中，分离、剪碎脊髓组织，置离心管中，加10倍0.125%