树鼩脊髓星形胶质细胞的分离鉴定  

Isolation and Identification of Spinal Astrocytes from Tree Shrews

作　　者：王璇 王文广[1] 李娜[1] 袁园 张志成 孙晓梅[1] WANG Xuan;WANG Wen-guang;LI Na;YUAN Yuan;ZHANG Zhi-cheng;SUN Xiao-mei(Institute of Medical Biology,Chinese Academy of Medicine Sciences (CAMS) & Peking UnionMedical College (PUMC) Institute of Medical Biology Tree Shrew Germplasm Resource Center,Institute of Medical Biology,Chinese Academy of Medicine Sciences Experimental TreeShrew Standardization and Application Research Provincial Innovation Team,The Key Laboratoryof Yunnan Province for Ophthalmic Research and Disease Control,Kunming 650118,China)

机构地区：[1]中国医学科学院/北京协和医学院医学生物学研究所树鼩种质资源中心中国医学科学院医学生物学研究所实验树鼩标准化与应用研究省创新团队云南省眼科疾病防治研究重点实验室

出　　处：《实验动物与比较医学》2019年第1期15-20,共6页Laboratory Animal and Comparative Medicine

基　　金：云南省应用基础研究计划面上项目(2018FB045);云南省科技人才和平台计划项目(2017HC019);重点实验室运行补助专项(2017DG008)

摘　　要：目的建立树鼩脊髓来源的星形胶质细胞的体外分离、鉴定方法以及原代培养技术。为体外利用树鼩星形胶质细胞开展相关研究提供实验材料。方法给新生树鼩实施安死术后采集脊髓组织,于解剖显微镜下去除脊髓膜和血管,减少成纤维细胞、血管内皮细胞和红细胞的影响,然后使用胰蛋白酶和DNaseⅠ联合消化眷髓组织,分离培养。根据星形胶质细胞和成纤维细胞、小胶质细胞的贴壁时间差异,使用差速贴壁法去除成纤维细胞;然后在细胞铺满后,使用恒温摇床振荡的方式去除少突胶质细胞;通过连续3次纯化,去除神经元。对所培养的细胞进行胶质纤维酸性蛋白(GFAP)细胞免疫荧光染色鉴定。结果采用胰蛋白酶和DNaseⅠ联合消化后差速贴壁培养,能够分离获得星形胶质细胞。细胞具有典型的原浆型和纤维型特征,原浆型呈多角状,突起少而粗壮;纤维型突起丰富且向外铺展开;细胞免疫荧光结果显示GFAP表达阳性,且纯度可达到95%。结论成功建立了树鼩的脊髓星形胶质细胞的体外分离纯化培养方法。Objective To establish an effective method of isolation,identification and primary culture method for astrocytes derived from the spinal cord of tree shrews in vitro,providing experimental materials for in vitro studies of spinal astrocytes.Methods Neonatal tree shrews were euthanized to collect spinal cord tissue,the spinal cord membrane and blood vessels were removed under a dissecting microscope to reduce the effects of fibroblasts,vascular endothelial cells and red blood cells.Then,the spinal cord tissues were digested with trypsin and DNase I,and cultured.According to the difference in the adherence time of astrocytes,fibroblasts and microglia,the fibroblasts were removed by differential adherence speed.After the cells were confluent,the non-staple gel was removed by shaking with a constant temperature shaker.Neurons were removed by three consecutive purifications.The cultured cells were identified by immunofluorescence staining of glial fibrillary acidic protein(GFAP).Results The astrocytes were isolated by differential adherent culture after trypsin and DNasel digestion.The isolated cells had typical protoplasmic and fibrous characteristics,the protoplasmic type was polygonal,the protrusions were few and strong,the fibrous protrusions are abundant and spread outward.The cellular immunofluorescence results showed that GFAP expression is positive,and the purity could reach 95%.Conclusion The isolation and purification of spinal cord astrocytes from tree shrews are successfully established.

关 键 词：星形胶质细胞 脊髓 分离鉴定 树鼩 

分 类 号：Q95-33[生物学—动物学]