基于p38MAPK信号因子重组甲型流感病毒M1/2诱导气管上皮细胞产生γ干扰素的研究

Based on p38MAPK signaling,recombinant influenza A virus proteins M1 and 2 induced tracheal epithelial cells to produce interferon-gamma

目的探讨重组甲型流感病毒基质蛋白1/2(rM1/2)诱导气管上皮细胞产生干扰素-γ的作用,以及基于p38MAPK信号因子的诱导作用机制。方法将小鼠原代气管上皮细胞分成6组,分别为M1组、M2组、病毒组、M1+病毒组、M2+病毒组、正常对照组。各组分别用相应制剂干预细胞4、8、24h,抑制试验中各实验组提前1h分别加入p38抑制剂,再进行相应制剂干预。提取细胞总RNA和总蛋白,分别采用RT-PCR和Western blot方法检测IFN-γmRNA和IFN-γ、p38MAPK、P-p38MAPK的表达。结果重组甲型流感病毒M1/2作用于小鼠气管上皮细胞,作用4h,8h,24h后的半定量RT-PCR和Western blot实验结果为rM1、rM2组诱导IFN-γmRNA表达量高于正常组,表示M1/2能诱导IFN-γ的产生。Western blot结果显示rM1、rM2组诱导P-p38MAPK表达量高于正常组,用p38MAPK抑制剂SB203580后,rM1联合病毒、rM2联合病毒组诱导P-p38MAPK表达量低于病毒组,且rM1联合病毒、rM2联合病毒组诱导IFN-γmRNA表达量低于病毒组,表示M1/2能诱导p38MAPK磷酸化。结论甲型流感病毒rM1/2能够在早期诱导小鼠气管上皮细胞产生IFN-γ;该作用与p38MAPK信号因子激活有关。

Objectives To investigate how matrix proteins 1 and 2 of a recombinant influenza A virus induce tracheal epithelial cells to produce interferon-γ and to investigate the mechanism of that induction based on p38MAPK signaling. Methods Mouse primary tracheal epithelial cells were divided into 6 groups, including （1） cells administered M1,（2）cells administered M2, （3）cells administered the virus, （4）cells administered M1 ＋the virus,（5）cells administered M2＋the virus, and （6） the control. Each group was treated with the corresponding treatment for 4 h, 8 h, and 24 h in the inhibition test. Experimental groups were treated with p38 inhibitor 1 h before being treated with the corresponding treatment. Total RNA and protein were extracted. The expression of IFN-γ mRNA and the expression of IFN-γ, p38MAPK, and P- p38MAPK proteins were detected with RT-PCR and Western blotting. Results Recombinant influenza A virus proteins M1 and 2 acted on mouse tracheal epithelial cells for 4 h, 8 h, or 24 h. RT-PCR and Western blotting indicated that the expression of IFN-γ mRNA was higher in cells treated with rM1 and rM2 group than in normal cells, so M1 and 2 can induce IFN-γ production. Western blotting indicated that the expression of p-p38MAPK was higher in cells treated with rM1 and rM2 than in normal cells. After cells were treated with the p38MAPK inhibitor SB203580, treatment in the form of rM1 and the virus or rM2 and the virus induced a higher level of P-p38MAPK expression than that in cells treated with the virus alone. Treatment in the form of rM1 and the virus or rM2 and the virus induced a lower level of IFN-γ mRNA expression that in cells treated with the virus alone, indicating that M1 and 2 can induce p38MAPK phosphorylation. Conclusion Influenza A virus rM1 and 2 can initially induce mouse tracheal epithelial cells to produce IFN-γ. This action is related to the activation of MAPK p38 signaling.