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目的:观察甲型流感病毒H3N2感染人胚肾上皮细胞(HEK-293T)细胞及人肺腺癌上皮细胞(A549)细胞后,对NF-κB转录活性以及TLR7、TNF-α、IL-8、IFN-βmRNA的影响,探讨H3N2对体外感染细胞中TLR7信号通路的影响及清热解毒中药毒热平注射液的干预作用。方法:采用MTT法体外检测各组药物对293T、A549细胞增殖的影响;H3N2感染293T细胞后,利用双荧光素酶报告系统,以利巴韦林、清开灵注射液为药物对照,检测毒热平注射液各浓度组细胞中NF-κB相对荧光素酶活性;H3N2感染A549细胞后,RT-PCR方法检测各组细胞中TLR7、TNF-α、IL-8、IFN-βmRNA水平。结果:MTT结果显示,毒热平、利巴韦林、清开灵注射液各浓度不影响细胞正常增殖(P>0.05)。报告基因结果显示,与细胞对照组相比,H3N2感染组细胞NF-κB转录活性显著增高(P<0.01);与H3N2感染组相比,利巴韦林0.5μg/mL,清开灵1/128稀释组,毒热平1μg/mL,10μg/mL,100μg/mL组均可不同程度下调NF-κB的转录活性(P<0.01)。RT-PCR结果显示,与细胞对照组比较,H3N2病毒组的TLR7、TNF-α、IL-8、IFN-βmRNA表达明显增高(P<0.01);与H3N2感染组相比,一定浓度的药物组TLR7、TNF-α、IL-8、IFN-βmRNA表达量明显降低(P<0.05或P<0.01)。结论:流感病毒H3N2攻击细胞可以显著上调TLR7受体的表达和核因子NF-κB转录活性,并引起下游相关靶基因的表达增多。毒热平注射液可以下调H3N2引起的TLR7信号通路的激活,抑制NF-κB的转录活性,减少下游炎性因子TNF-α、IL-8、IFN-β的表达。关键词:H3N2;毒热平注射液;NF-κB;双荧光素酶报告系统;TLR7;信号转导
OBJECTIVE: To observe the transcriptional activity of NF-κB and the expressions of TLR7, TNF-α, IL-8 and TNF-α in human embryonic kidney epithelial cells (HEK-293T) and human lung adenocarcinoma epithelial cells (A549) IFN-βmRNA, to explore the effect of H3N2 on TLR7 signaling pathway in vitro and the intervention effect of Qingrejiedudududu injection. Methods: MTT assay was used to detect the effect of each drug on the proliferation of 293T and A549 cells. After 293N cells were infected by H3N2, the dual luciferase reporter system and ribavirin and Qingkailing injection were used as the drug control, The level of TLR7, TNF-α, IL-8 and IFN-βmRNA in each group were detected by RT-PCR after A549 cells were infected with H3N2. Results: The results of MTT showed that the concentrations of Tachien-Ping, Ribavirin and Qingkailing Injection did not affect the normal proliferation of cells (P> 0.05). The results of reporter gene showed that the transcriptional activity of NF-κB in H3N2 infected group was significantly higher than that in control group (P <0.01). Compared with H3N2 infected group, ribavirin 0.5 μg / mL, Qingkailing 1 / 128 dilution group, TDP 1μg / mL, 10μg / mL and 100μg / mL groups could down-regulate the transcriptional activity of NF-κB to some extent (P <0.01). RT-PCR results showed that the expression of TLR7, TNF-α, IL-8 and IFN-βmRNA in H3N2 virus group was significantly higher than that in control group (P <0.01). Compared with H3N2-infected group, The expression of TLR7, TNF-α, IL-8 and IFN-β mRNA were significantly decreased (P <0.05 or P <0.01). Conclusion: Influenza virus H3N2 can up-regulate the expression of TLR7 receptor and nuclear factor NF-κB transcriptional activity and induce the expression of downstream target genes. TDP can down-regulate the activation of TLR7 signaling pathway induced by H3N2, inhibit the transcriptional activity of NF-κB and decrease the expression of downstream inflammatory cytokines TNF-α, IL-8 and IFN-β. Keywords: H3N2; Toxin Ping Injection; NF-κB; Dual-luciferase reporter system; TLR7; Signal transduction