Pyrin在巨噬细胞M1型极化中的作用研究

Role of Pyrin in macrophage M1 polarization

摘要:

目的：探讨Pyrin在巨噬细胞M1型极化中的作用。方法：培养人单核细胞株THP-1细胞，提取人外周血单核细胞（peripheral blood mononuclear cell，PBMC）、小鼠骨髓来源巨噬细胞（bone marrow-derived macrophage，BMDM）。随机分为对照组和实验组，实验组用脂多糖（lipopolysaccharide，LPS，100 ng/mL）复合干扰素-γ（interferon-γ，IFN-γ，20 ng/mL）干预（THP-1 18 h，PBMC、BMDM 24 h）构建巨噬细胞M1型极化模型。qRT-PCR、Western blot和流式细胞术检测2组M1型极化相关标志物，同时利用qRT-PCR和Western blot检测MEFV基因和Pyrin蛋白表达水平。结果：与对照组相比，实验组在LPS+IFN-γ刺激后，qRT-PCR检测THP-1细胞M1型极化标志物：肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）（t=-4.360，P=0.007）、白细胞介素-1β（interleukin-1β，IL-1β）（t=-8.329，P=0.000）、白细胞介素-6（interleukin-6，IL-6）（t=-2.924，P=0.033）、CD14（t=-2.858，P=0.035）、CD80（t=-3.433，P=0.019），PBMC细胞M1型极化标志物：TNF-α（t=-2.893，P=0.034）、IL-1β（t=-3.606，P=0.015）、IL-6（t=-2.895，P=0.034）、CD14（t=-2.645，P=0.046）、CD80（t=-3.648，P=0.015），BMDM细胞M1型极化标志物：TNF-α（t=-6.123，P=0.002）、IL-1β（t=-2.697，P=0.043）、单核细胞趋化蛋白-1（monocyte chemotactic protein 1，MCP-1）（t=-4.335，P=0.007）、诱导型一氧化氮合酶（inducible nitric oxide synthase，iNOS）（t=-3.607，P=0.015）均明显增加，Western blot检测THP-1细胞M1型极化标志物：TNF-α（t=-3.827，P=0.031）、IL-1β（t=-4.189，P=0.025）、IL-6（t=-5.345，P=0.002）均明显增加，流式细胞术检测THP-1细胞M1型极化标志物HLA-DR（t=-3.270，P=0.017）、BMDM细胞M1型极化标志物F4/80+CD11c+（t=-3.833，P=0.009）均明显增加，提示M1型极化模型构建成功；在构建成功的M1型极化模型中，qRT-PCR检测THP-1细胞、PBMC细胞和BMDM细胞MEFV基因表达水平较对照组明显增加（t=-3.226，P=0.023；t=-2.989，P=0.030；t=-2.891，P=0.034），Western blot检测THP-1细胞Pyrin蛋白表达水平较对照组明显增加（t=-8.591，P=0.000）。结论：巨噬细胞M1型极化中Pyrin表达增加，Pyrin可能与巨噬细胞M1型极化相关。

Abstract:

Objective：To investigate the role of Pyrin in macrophage M1 polarization. Methods：THP-1 cells were cultured and periph－eral blood mononuclear cell（PBMC），bone marrow-derived macrophage（BMDM） were extracted. These three kinds of cells were ran－domly divided into control group and treatment group. Treatment group was treated with lipopolysaccharide（LPS，100 ng/mL） and in－terferon-γ（IFN-γ，20 ng/mL） to construct macrophage M1 polarization models（THP-1 for 18 h，PBMC and BMDM for 24 h）. qRT-PCR，Western blot and flow cytometry（FCM） were used to test the markers of M1 polarization，qRT-PCR and Western blot were used to test the expression of MEFV and Pyrin. Results：Compared with those of control group，qRT-PCR detection of THP-1-M1 polarization markers：TNF-α（t=-4.360，P=0.007），IL-1β（t=-8.329，P=0.000），IL-6（t=-2.924，P=0.033），CD14（t=-2.858，P=0.035），CD80（t=-3.433，P=0.019） and Western blot detection of THP-1-M1 polarization markers：TNF-α（t= -3.827，P=0.031），IL-1β（t=-4.189，P=0.025），IL-6（t=-5.345，P=0.002） were significantly increased after the treatment with LPS and IFN-γ. The mRNA levels of TNF-α（t=-2.893，P=0.034），IL-1β（t=-3.606，P=0.015），IL-6（t=-2.895，P=0.034），CD14（t=-2.645，P=0.046），CD80（t=-3.648，P=0.015） of PBMC-M1 polarization markers and the mRNA levels of TNF-α（t=-6.123，P=0.002），IL-1β（t=-2.697，P=0.043），MCP-1（t=-4.335，P=0.007），iNOS（t=-3.607，P=0.015） of BMDM-M1 polarization markers were signifi－cant higher after the treatment with LPS and IFN-γ. Treatment group displayed higher positive cells of THP-1-M1 polarization marker HLA-DR（t=-3.270，P=0.017） and BMDM-M1 polarization marker F4/80+CD11c+（t=-3.833，P=0.009） than control group. These results suggested that the M1 polarization models were constructed successfully. In these three M1 polarization models，qRT-PCR and Western blot detection of MEFV（t=-3.226，P=0.023） and Pyrin（t=-8.591，P=0.000） of THP-1-M1 macrophages were significantly increased. The mRNA level of MEFV of PBMC-M1 macrphages（t=-2.989，P=0.030） and BMDM-M1 macrophages（t=-2.891，P=0.034） were significant higher than control group. Conclusion：The expressions of Pyrin increase in macrophage M1 polariza－tion；Pyrin maybe related to macrophage M1 polarization.