在体外感染 VII 型新城疫病毒后鸡外周血单核细胞细胞外基质降解和迁移的检测。
Assay of extracellular matrix degradation and transmigration of chicken peripheral blood mononuclear cells after infection with genotype VII Newcastle disease virus in vitro.
机构信息
Animal Infectious Disease Laboratory, School of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China; Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonosis, Yangzhou University, Yangzhou 225009, China; Key Laboratory of Prevention and Control of Biological Hazard Factors (Animal Origin) for Agri-Food Safety and Quality, Ministry of Agriculture of China, Yangzhou University, Yangzhou 225009, China.
Animal Infectious Disease Laboratory, School of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China; Joint International Research Laboratory of Agriculture and Agri-Product Safety, The Ministry of Education of China, Yangzhou University, Yangzhou 225009, China; Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonosis, Yangzhou University, Yangzhou 225009, China.
出版信息
J Virol Methods. 2021 Apr;290:114076. doi: 10.1016/j.jviromet.2021.114076. Epub 2021 Jan 27.
Previous studies showed that, compared to genotype IV Newcastle disease virus (NDV), genotype VII NDV induced extensive extracellular matrix (ECM) degradation by up-regulating the protein expression of matrix metalloproteinase (MMP)-14 in chicken spleens. To investigate potential relationship between MMP-14 function and the ECM degradation, an in vitro peripheral blood mononuclear cells (PBMCs) infection model was established to study the effect of genotype VII NDV (JS5/05) infection on MMP-14 expression, ECM degradation and cell transmigration. The gene and protein expression levels of MMP-14 in NDV-infected chicken PBMCs were measured by quantitative real-time PCR (qRT-PCR) and Western blot, and the subcellular location of MMP-14 was analyzed using immunofluorescence microscopy. A fluorescence-based collagen degradation assay was optimized to measure ECM degradation in PBMCs. Additionally, parameters of a transwell-based transmigration assay were also optimized to determine chemotaxis and transmigration of virus-infected PBMCs. The results showed that JS5/05 up-regulated significantly the expression of MMP-14 in PBMCs at the mRNA and protein levels compared to genotype IV NDV (Herts/33). MMP-14 was transported towards the membrane and accumulated on the cell surface of the JS5/05-infected cells, whereas it remained mainly in the cytoplasm of the Herts/33-infected cells. Collagen degradation assay showed that JS5/05-infected cells exhibited significant collagen degradation compared to the Herts/33-infected cells, and the areas of collagen degradation co-localized with cell surface MMP-14 in the JS5/05-infected cells. The transwell-based transmigration system showed that the transmigration of the JS5/05-infected PBMCs was enhanced significantly compared to the Herts/33-infected cells. These results demonstrated that genotype VII NDV induced up-regulation and surface accumulation of MMP-14 in PBMCs, leading to enhanced ECM degradation and cell migration, and the assays optimized for this study were useful for investigating the regulation of cell behaviour by NDV.
先前的研究表明，与基因型 IV 新城疫病毒（NDV）相比，基因型 VII NDV 通过上调鸡脾脏中基质金属蛋白酶（MMP）-14 的蛋白表达，诱导广泛的细胞外基质（ECM）降解。为了研究 MMP-14 功能与 ECM 降解之间的潜在关系，建立了体外外周血单核细胞（PBMC）感染模型，以研究基因型 VII NDV（JS5/05）感染对 MMP-14 表达、ECM 降解和细胞迁移的影响。通过定量实时 PCR（qRT-PCR）和 Western blot 测量 NDV 感染鸡 PBMC 中 MMP-14 的基因和蛋白表达水平，并通过免疫荧光显微镜分析 MMP-14 的亚细胞定位。优化了基于荧光的胶原降解测定法来测量 PBMC 中的 ECM 降解。此外，还优化了基于 Transwell 的迁移测定法的参数，以确定病毒感染的 PBMC 的趋化性和迁移性。结果表明，与基因型 IV NDV（Herts/33）相比，JS5/05 显著上调了 PBMC 中 MMP-14 的表达，无论是在 mRNA 还是蛋白水平。MMP-14 被转运到细胞膜上，并在 JS5/05 感染细胞的细胞表面上积累，而在 Herts/33 感染细胞中，它主要留在细胞质中。胶原降解测定表明，与 Herts/33 感染细胞相比，JS5/05 感染细胞表现出明显的胶原降解，并且在 JS5/05 感染细胞中，胶原降解区域与细胞表面 MMP-14 共定位。基于 Transwell 的迁移系统显示，与 Herts/33 感染细胞相比，JS5/05 感染的 PBMC 的迁移明显增强。这些结果表明，基因型 VII NDV 诱导 PBMC 中 MMP-14 的上调和表面积累，导致 ECM 降解和细胞迁移增强，本研究优化的测定法可用于研究 NDV 对细胞行为的调节。
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