树鼩脊髓微血管内皮细胞的分离培养及EV71感染实验

doi:10.12300/j.issn.1674-5817.2020.208

实验动物与比较医学 ›› 2021, Vol. 41 ›› Issue (2): 148-154.DOI: 10.12300/j.issn.1674-5817.2020.208

所属专题：实验动物资源开发与利用

树鼩脊髓微血管内皮细胞的分离培养及EV71感染实验

施梅言, 王璇, 王文广, 阮蕾颖, 代解杰

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,昆明 650118

收稿日期:2020-12-14 修回日期:2021-03-11 出版日期:2021-04-25 发布日期:2021-04-30
作者简介:施梅言(1994—), 女, 硕士研究生, 研究方向: 人类疾病动物模型建立及相关研究。E-mail: 1758651731@qq.com
基金资助:
云南省应用基础研究计划项目(2018FB045); NSFC-云南联合基金重点支持项目(U1702282); 云南省科技人才和平台计划项目(2017HC019); 昆明市科技创新团队(2019-1-R-24483)

Isolation and Culture of Spinal Microvascular Endothelial Cells of Tree Shrews and Experimental Study on Infection with Enterovirus 71

SHI Meiyan, WANG Xuan, WANG Wenguang, RUAN Leiying, DAI Jiejie

Tree Shrew Germplasm Resource Center, Institute of Medical Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Kunming 650118, China

Received:2020-12-14 Revised:2021-03-11 Online:2021-04-25 Published:2021-04-30

摘要/Abstract

摘要： 目的建立树鼩脊髓来源的微血管内皮细胞体外分离培养技术,用71型肠道病毒（enterovirus 71,EV71）感染该细胞,探讨其感染特性,为EV71损伤中枢神经系统机制的研究提供基础。方法采用Ⅱ型胶原酶、分散酶、DNase Ⅰ三酶联合两次消化树鼩脊髓组织后,获取微血管内皮细胞,并进行纯化和鉴定。用EV71感染树鼩脊髓微血管内皮细胞,测定感染不同时间点的细胞及其培养上清液中病毒载量。采用间接免疫荧光法检测细胞中EV71标志蛋白的表达,以确定EV71对树鼩脊髓微血管内皮细胞的感染性。结果分离培养获得的树鼩脊髓微血管内皮细胞呈典型的分支状和串珠状,经过嘌呤霉素纯化培养后的传代细胞主要是不规则的多角状细胞;细胞免疫荧光鉴定结果显示,微血管内皮细胞标志蛋白CD31和血管性血友病因子（von Willebrand factor,vWF）表达都呈阳性。用感染复数为1的EV71感染树鼩脊髓微血管内皮细胞,可见典型的细胞病变,且病毒滴度可达3.2×10⁶ TCID₅₀/mL,表明EV71可成功感染树鼩脊髓微血管内皮细胞并有效增殖;而且在48h内,细胞培养上清液中病毒载量呈线性升高,而细胞中病毒载量在12h到达顶峰。间接免疫荧光法在EV71感染后12 h的树鼩脊髓微血管内皮细胞质中可检测到病毒颗粒。结论成功建立了树鼩脊髓微血管内皮细胞体外分离培养方法,确定了EV71对树鼩脊髓微血管内皮细胞的感染性和病毒增殖特性,为开展EV71入侵中枢神经系统的机制研究提供了一定基础。

关键词: 脊髓微血管内皮细胞, 分离培养, EV71, 病毒增殖, 树鼩

Abstract: Objective To establish an effective method of isolation and primary culture method for microvascular endothelial cells derived from the spinal cord of tree shrews in vitro. Enterovirus 71 (EV71) was used to infect these cells to explore its infectious characteristics and provide a reference for the study of the mechanism of EV71 induced damage to the central nervous system. Methods The spinal cord tissues were digested twice with type Ⅱ collagenase, dispase, and DNaseⅠ, then the microvascular endothelial cells were obtained. EV71 was used to infect tree shrew spinal microvascular endothelial cells, and the virus titer at different time points was measured. The expression of EV71 in the infected cells was detected by immunofluorescence assay to determine the infectivity of EV71 to the spinal cord microvascular endothelial cells of tree shrews. Results The microvascular endothelial cells were typically branched and beaded, and the passage cells obtained after puromycin purification and culture were mainly irregular polygonal cells. The cellular immunofluorescence results showed that CD31 and vWF expressions were positive. The spinal microvascular endothelial cells of tree shrews were infected with EV71 at a multiplicity of infection of 1, the cells showed typical cytopathic appearance, and the virus titer was approximately 3.2×10⁶ TCID₅₀/mL. This proved that EV71 infected and proliferated in the tree shrew spinal cord microvascular endothelial cells, and within 48 h, the viral load in the supernatant increased linearly, reaching its peak at 12 h. The indirect immunofluorescence method detected virus particles in the cytoplasm of the cells 12 h after infection. Conclusions The isolation and purification methods of spinal cord microvascular endothelial cells from tree shrews are successfully established, and the infectivity to the obtained cells and proliferation of EV71 in the cells are confirmed, providing a basis for the study of the mechanism of EV71 invading the central nervous system.

Key words: Spinal cord microvascular endothelial cells, Isolation and culture, Enterovirus 71, Virus proliferation, Tree shrews

中图分类号:

R-332
Q95-33

施梅言, 王璇, 王文广, 阮蕾颖, 代解杰. 树鼩脊髓微血管内皮细胞的分离培养及EV71感染实验[J]. 实验动物与比较医学, 2021, 41(2): 148-154.

SHI Meiyan, WANG Xuan, WANG Wenguang, RUAN Leiying, DAI Jiejie. Isolation and Culture of Spinal Microvascular Endothelial Cells of Tree Shrews and Experimental Study on Infection with Enterovirus 71[J]. Laboratory Animal and Comparative Medicine, 2021, 41(2): 148-154.

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树鼩脊髓微血管内皮细胞的分离培养及EV71感染实验

Isolation and Culture of Spinal Microvascular Endothelial Cells of Tree Shrews and Experimental Study on Infection with Enterovirus 71

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