| Dengue virus(DENV) is one kind of arbovirus Aedes aegypti and Aedes albopictus are the major mosquito vectors. Approximately 5-5.28 billion people are infected with dengue virus in every year. Dengue virus infection cause dengue fever, and severe dengue haemorrhagic fever/dengue shock syndrome(DHF/DSS), which is characterized by systemic capillary leakage. It is generally viewed that DHF/DSS is associated with toxicity of virus, Ab-dependent enhancement(ADE), host, T cell cross-immune reaction and cytokines storming. Numerous research mechanism raised because the model used is different, the current model in mice and human vascular endothelial cells in particular of human umbilical vein endothelial cells are the main pathogenesis of DENV infection model, and vascular endothelial cells is one of the important targets of DHF/DSS.But mouse models can’t completely model the symptom of infection with human, and the effect factors of the single cell m o d e l c a n ’ t e x a c t l y r e f l e c t t h e s y m p t o m s o f i n f e c t i o n.Recent studies have reported that mechanical stimulus can affect cellular behavior. Vascular basement membrane and the lower substrate elastic hardness modulus played an important role in vascular endothelial cellular functions and behaviors,including survival, breeding, migration, maintain steady state, and these biological functions under the conditions of different elastic hardness level are differenced. Nowadays, it uses of polyacrylamide gel stiffness organizing different hardness, which can affect the biological function of the cell, making it more close to the physical environment. The successful establishment of hydrogel substrates as the model of vascular basement membranes might provide a new way for the investigation of the pathogenesis of DENV infection.Objective To analyze the effects of dengue virus 2(DENV-2) infection on the expression of IL-29 in primary human umbilical vein endothelial cell(HUVECs) cultured on hydrogel substrates. Methods 1.HUVEC culture and identification:Primary HUVECs were separated from fresh human umbilical vein and identified by using the Flow cytometry and immunocytochemistry.2.The preparation of hydrogels and cell inoculation:Primary HUVECs were isolated and cultured on hydrogel substrates.3.Flow cytometry detect the apoptosis and infection rate:DENV-2 stains were used to infect the primary HUVECs at a multiplicity of infection(MOI) of 10. Flow cytometry analysis was performed to detect the apoptosis and infection rate of HUVECs after 48 hours of culturing. 4.The experiment of gene chips:The gene chip profiling was performed to analyze m RNA expression. 5.Realtime PCR and double antibody sandwich ELISA assay:The expression of IL-29 at m RNA and protein levels were measured by real-time fluorescent quantitative PCR analysis and double antibody sandwich ELISA assay, respectively. Results Compared with 96.36% of baby hamster kidney(BHK) cells that were infected with DENV-2 stains, only 4.71% primary HUVECs cultured on hydrogel substrates were infected. The primary HUVECs cultured on hydrogel substrates with or without DENV-2 infection showed no significant differences with the rates of cell apoptosis and infection(P>0.05). A significant difference was observed with the expression of IL-29 at m RNA level between primary HUVECs cultured on hydrogel substrates and the cells cultured in plastic bottles(P<0.05). The results of the real-time quantitative PCR analysis and ELISA assay showed that IL-29 was highly expressed in DENV-2 infected primary HUVECs cultured on hydrogel substrates as compared with those in control groups(P<0.05). Conclusion The expression of IL-29 was detected in DENV-2 infected primary HUVECs cultured on hydrogel substrates, which was significantly different from that in DENV-2 infected primary HUVECs cultured in plastic bottles.The successful establishment of hydrogel substrates as the model of vascular basement membranes might provide a new way for the investigation of the pathogenesis of DENV infection. |
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