树鼩脊髓星形胶质细胞的分离鉴定

doi:10.3969/j.issn.1674-5817.2019.01.004

实验动物与比较医学 ›› 2019, Vol. 39 ›› Issue (1): 15-20.DOI: 10.3969/j.issn.1674-5817.2019.01.004

树鼩脊髓星形胶质细胞的分离鉴定

王璇, 王文广, 李娜, 袁园, 张志成, 孙晓梅

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, 昆明 650118

收稿日期:2018-07-27 出版日期:2019-02-25 发布日期:2021-01-29
作者简介:王璇(1993-),男,硕士,研究方向:病原生物学。E-mail:w_wangxuan@outlook.com
基金资助:
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)

Isolation and Identification of Spinal Astrocytes from Tree Shrews

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 Union Medical College PUMC Institute of Medical Biology Tree Shrew Germplasm Resource Center,Institute of Medical Biology, Chinese Academy of Medicine Sciences Experimental Tree Shrew Standardization and Application Research Provincial Innovation Team, The Key Laboratory of Yunnan Province for Ophthalmic Research and Disease Control, Kunming 650118, China

Received:2018-07-27 Online:2019-02-25 Published:2021-01-29

摘要/Abstract

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联合消化脊髓组织,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。

关键词: 星形胶质细胞, 脊髓, 分离鉴定, 树鼩

Abstract: 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 DNaseI 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.

Key words: Astrocytes, Spinal cord, Isolation and identification, Tree shrews

中图分类号:

Q95-33

王璇, 王文广, 李娜, 袁园, 张志成, 孙晓梅. 树鼩脊髓星形胶质细胞的分离鉴定[J]. 实验动物与比较医学, 2019, 39(1): 15-20.

WANG Xuan, WANG Wen-guang, LI Na, YUAN Yuan, ZHANG Zhi-cheng, SUN Xiao-mei. Isolation and Identification of Spinal Astrocytes from Tree Shrews[J]. Laboratory Animal and Comparative Medicine, 2019, 39(1): 15-20.

参考文献

[1] Anna V, Robert K, Erik U, et al.Astrocytes and disease: a neurodevelopmental perspective[J]. Genes Dev, 2012, 26(9):891-907.
[2] Powell EM, Geller HM.Dissection of astrocyte-mediated cues in neuronal guidance and process extension[J]. Glia, 1999, 26(1):73-83.
[3] Nagele RG, Wegiel J, Venkataraman V, et al.Contribution of glial cells to the development of amyloid plaques in Alzheimer’s disease[J]. Neurobiol Aging, 2004, 25(5):663-674.
[4] Barcia C, Ros CM, Annese V, et al.IFN-α signaling, with the synergistic contribution of TNF-〈, mediates cell specific microglial and astroglial activation in experimental models of Parkinson’s disease[J]. Cell Death Dis, 2011, 2: e142.
[5] Librizzi L, Noe F, Vezzani A, et al.Seizure-induced brain-borne inflammation sustains seizure recurrence and blood-brain barrier damage[J]. Ann Neurol, 2012, 72(1):82-90.
[6] Molofsky AV, Krencik R, Ullian EM, et al.Astrocytes and disease: a neurodevelopmental perspective[J]. Genes Dev, 2012, 26(9):891-907.
[7] Kuhn TB.Growing and working with spinal motor neurons[J]. Methods Cell Biol, 2003, 71:67-87.
[8] Soula C, Danesin C, Kan P, et al.Distinct sites of origin of oligodendrocytes and somatic motoneurons in the chick spinal cord: oligodendrocytes arise from Nkx2.2-expressing progenitors by a Shh-dependent mechanism[J]. Development, 2001, 128(8):1369-1379.
[9] Villarreal A, Rosciszewski G, Murta V, et al.Isolation and characterization of ischemia-derived astrocytes (IDAs) with ability to transactivate quiescent astrocytes[J]. Front Cell Neurosci, 2016, 10:139.
[10] Beaudet MJ, Yang Q, Cadau S, et al.High yield extraction of pure spinal motor neurons, astrocytes and microglia from single embryo and adult mouse spinal cord[J]. Sci Rep, 2015, 5:16763.
[11] Pawlik M, Fuchs E, Walker LC, et al.Primate-like amyloidβ sequence but no cerebral amyloidosis in aged tree shrews[J]. Neurobiol Aging, 1999, 20(1):47-51.
[12] 祁可可, 冯敏, 孟肖路, 等. 树鼩的社会挫败抑郁模型[J].心理科学进展, 2012, 20:1787-1793.
[13] Yamashita A, Fuchs E, Taira M, et al.Somatostatin-immunoreactive senile plaque-like structures in the frontal cortex and nucleus accumbens of aged tree shrews and Japanese macaques[J]. J Med Primatol, 2012, 41:147-157.
[14] Walter E, Keist R, Niederöst B, et al.Hepatitis B virus infection of tupaia hepatocytes in vitro and in vivo[J]. Hepatology, 1996, 24(1):1-5.
[15] 庞其方, 万新邦, 胥爱源, 等. 乙型肝炎病毒( HBV) 感染树鼩的实验研究[J]. 医学研究通讯, 1981 (9):11-12.
[16] Feng Y, Feng YM, Lu C, et al.Tree shrew, a potential animal model for hepatitis C,supports the infection and replication of HCV in vitro and in vivo[J]. J Gen Virol, 2017, 98(8):2069-2078.
[17] Zhao X, Tang ZY, Klumpp B, et al.Primary hepatocytes of Tupaia belangeri as a potential model for hepatitis C virus infection[J]. J Clin Invest, 2002, 109(2):221-232.
[18] Li L, Li Z, Wang E, et al.Herpes simplex virus 1 infection of tree shrews differs from that of mice in the severity of acute infection and viral transcription in the peripheral nervous system[J]. J Virol, 2015, 90(2):790-804.
[19] Darai G, Schwaier A, Komitowski D, et al.Experimental infection of Tupaia belangeri (tree Shrews) with herpes simplex virus types 1 and 2[J]. J Infect Dis, 1978, 137(3):221-226.
[20] Yang ZF, Zhao J, Zhu YT, et al.The tree shrew provides a useful alternative model for the study of influenza H1N1 virus[J]. Virol J, 2013, 10:111.
[21] 王文广, 黄晓燕, 徐娟, 等. EV71 可感染幼龄中缅树鼩[J].动物学研究, 2012, 33(1):7-13.
[22] Wang J, Zhang Y, Zhang X, et al.Pathologic and immunologiccharacteristics of coxsackievirus A16 infection in rhesus macaques[J]. Virology, 2017, 500:198-208.
[23] Li JP, Liao Y, Zhang Y, et al.Experimental infection of treeshrews (Tupaia belangeri) with Coxsackie virus A16[J]. Zool Res, 2014, 35(6):485-491.
[24] 曾俊. 柯萨奇病毒感染星形胶质细胞及细胞因子变化研究[D]. 汕头: 汕头大学, 2010.
[25] 马娜. EV71对星形胶质细胞的感染及其病理意义的研究[D]. 昆明: 昆明医科大学, 2013.
[26] 蒋治武, 王革非, 周健祥, 等. 流感病毒感染小鼠星形胶质细胞诱导GDNF转录水平上调[J]. 中国热带医学, 2012,12(6):649-651.
[27] 黄睿. HCMV感染上调人星形胶质细胞IGF2分泌对胶质瘤细胞生物学行为影响的分子机制[D]. 青岛: 青岛大学, 2015.
[28] Ullian Em, Sappers tein SK, Chris topherson KS, et al. Control of synapse number by glia[J]. Science, 2001, 291(5504):657-661.
[29] Gingras M, Gagnon V, Minotti S, et al.Optimized protocols for isolation of primary motor neuronsastrocytes and microglia from embryonic mouse spinal cord[J]. J Neurosci Methods, 2007, 163(1):111-118.
[30] 马超, 冯世庆, 班德翔, 等. 大鼠脊髓星形胶质细胞的体外培养与鉴定[J]. 中国医学创新, 2014, 11(2):9-10.
[31] McCarthy KD, de Vellis J. Preparation of separate as troglial and oligodendroglial cell cultures from rat cerebral tissue[J]. Cell Biol, 1980, 85(3):890-902.
[32] Gerhardt H, Golding M, Fruttiger M, et al.VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia[J]. Cell Biol, 2004, 161(6):1163-1177.
[33] 黄太萍, 黄巨恩, 沈丽. 体外原代星形胶质细胞分离培养方法的优化[J]. 广西医科大学学报, 2014, 31(6):894-897.

树鼩脊髓星形胶质细胞的分离鉴定

Isolation and Identification of Spinal Astrocytes from Tree Shrews

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