糖蛋白M6B对RAW264.7巨噬细胞炎症因子表达的影响

张顺; 杨容金; 张飞宇; 张晓萌; 常小倩; 邢萍萍; 阮班军; 王姗; 陶凌

doi:10.12125/j.chj.202109071

糖蛋白M6B对RAW264.7巨噬细胞炎症因子表达的影响

Effects of glycoprotein M6B on expression of RAW264.7 macrophages inflammatory factors

摘要

摘要:
目的研究糖蛋白M6B（glycoprotein M6B，GPM6B）对巨噬细胞炎症因子IL-1β、TNF-α、IFN-γ和IL-6的影响。
方法 ①Western blot和qPCR检测RAW264.7经典激活的巨噬细胞（classically activated macrophage, M1）和替代激活的巨噬细胞（alternatively activated macrophage, M2）中GPM6B表达量变化。②分别用对照组（sh-Scramble）和GPM6B干涉组（sh-GPM6B1和sh-GPM6B2）的慢病毒感染RAW264.7巨噬细胞系，荧光显微镜下观察病毒的感染效率。Western blot和qPCR检测GPM6B的干涉效率。③qPCR检测对照组和GPM6B干涉组促炎因子IL-1β、TNF-α和IFN-γ的mRNA表达水平，ELISA法检测上清中IL-1β和IL-6的含量。④Western blot检测干涉GPM6B后炎症相关分子p65和Iκα的磷酸化水平变化。
结果 ①相比于对照组，RAW264.7巨噬细胞M1型中GPM6B蛋白和mRNA水平均显著升高（P<0.05），而M2型中GPM6B蛋白和mRNA水平均显著降低（P<0.05）。②与sh-Scramble组相比，sh-GPM6B1组和sh-GPM6B2组GPM6B蛋白和mRNA水平均显著降低（P<0.05）。③干涉GPM6B后促炎因子IL-1β、TNF-α和IFN-γ的mRNA表达水平和上清中IL-1β和IL-6均显著降低（P<0.05）。④干涉GPM6B后炎症相关分子p65和Iκα的磷酸化水平降低。
结论干涉GPM6B可抑制RAW264.7巨噬细胞促炎因子的表达。

Abstract:
AIM To investigate the role of Glycoprotein M6B (GPM6B) on the production of macrophage inflammatory cytokines, including IL-1β, TNF-α, IFN-γ and IL-6.
METHODS Western blot and qPCR were used to detect GPM6B expression in RAW264.7 macrophages classically activated M1 and alternatively activated M2. RAW264.7 macrophage lines were infected with lentivirus of the control group (sh-Scramble) and the knockdown GPM6B group (sh-GPM6B1 and sh-GPM6B2) respectively, and the infection efficiency of the virus was observed under fluorescence microscope and confirmed by Western blot and qPCR. The mRNA expression levels of pro-inflammatory factors IL-1β, TNF-α and IFN-γ in the control group and the GPM6B intervention group were detected by qPCR, and the contents of IL-1β and IL-6 in supernatant were detected by ELISA. The phosphorylation levels of inflammatory molecules p65 and Iκα were detected by Western blot after GPM6B interference.

RESULTS Compared with those in the control group, the protein and mRNA levels of GPM6B in RAW264.7 macrophages M1 type were significantly increased, while in M2 they were significantly decreased (P<0.05). Compared with those in the sh-Scramble group, the interference efficiency of GPM6B in the sh-GPM6B1 and sh-GPM6B2 groups was significantly decreased in protein and mRNA levels (P<0.05). The expression of pro-inflammatory factors mRNA, including IL-1β, TNF-α and IFN-γ, and the secretion levels of IL-1β and IL-4 in supernatant were significantly decreased after GPM6B knockdown (P<0.05). The phosphorylation levels of inflammatory molecules P65 and Iκα decreased after GPM6B intervention (P<0.05).
CONCLUSION Interference with GPM6B inhibits the expression of proinflammatory factors released by RAW264.7 macrophages.

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