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

doi:10.12300/j.issn.1674-5817.2020.208

Abstract

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

CLC Number:

R-332
Q95-33

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.

Figures/Tables 5

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