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为初步探索EV-A71在小鼠巨噬细胞中的复制情况和抗病毒的固有免疫应答,本文以小鼠巨噬细胞系RAW264.7为细胞模型,通过建立EV-A71绝对定量qPCR方法检测EV-A71病毒载量;EV-A71和紫外灭活的EV-A71感染RAW264.7,不同时间点提取总RNA,RT-qPCR检测促炎细胞因子、趋化因子和模式识别受体的mRNA表达变化水平。本研究成功建立了EV-A71的绝对定量qPCR检测方法,并发现EV-A71感染RAW264.7后随着感染时间的延长,EV-A71病毒载量呈递减趋势;EV-A71和紫外灭活的EV-A71可以引起IL-1β、IL-6、TNF-α促炎细胞因子和IP-10、MCP-1、MIP-1α趋化因子反应,上调TLR2、TLR1、TLR6、MDA5和RIG-I mRNA表达。研究结果显示,EV-A71在小鼠巨噬细胞中具有较低水平的复制,同时产生促炎细胞因子和趋化因子反应。
To initially explore the EV-A71 replication in mouse macrophages and antiviral innate immune response, the mouse macrophage cell line RAW264.7 as a cell model, by establishing EV-A71 absolute quantitative qPCR method to detect EV -A71 viral load; RAW264.7 was infected with EV-A71 and UV-inactivated EV-A71, and the total RNA was extracted at different time points. The mRNA expression of pro-inflammatory cytokines, chemokines and pattern recognition receptors was detected by RT-qPCR Level. In this study, the absolute quantitative qPCR assay of EV-A71 was successfully established and found that the EV-A71 viral load showed a decreasing trend with the prolongation of infection time after RAW264.7 infection with EV-A71. The EV-A71 and UV-inactivated EV-A71 can induce the chemokine response of IL-1β, IL-6, TNF-α pro-inflammatory cytokines and IP-10, MCP-1 and MIP-1α chemokines and upregulate the expressions of TLR2, TLR1, TLR6, MDA5 and RIG-I mRNA expression. The results show that EV-A71 has a lower level of replication in mouse macrophages, with production of proinflammatory cytokines and chemokine responses.