[1]贾康,张志,刘昌玲,等.神经调节素受体降解蛋白1调控巨噬细胞极化对成纤维细胞功能的影响[J].第三军医大学学报,2021,43(11):1003-1009.
 JIA Kang,ZHANG Zhi,LIU Changling,et al.Effect of neuregulin receptor degradation protein 1 regulation of macrophage polarization on function of fibroblasts in vitro[J].J Third Mil Med Univ,2021,43(11):1003-1009.
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神经调节素受体降解蛋白1调控巨噬细胞极化对成纤维细胞功能的影响(/HTML )
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《第三军医大学学报》[ISSN:1000-5404/CN:51-1095/R]

卷:
43卷
期数:
2021年第11期
页码:
1003-1009
栏目:
专题报道
出版日期:
2021-06-15

文章信息/Info

Title:
Effect of neuregulin receptor degradation protein 1 regulation of macrophage polarization on function of fibroblasts in vitro
作者:
贾康张志刘昌玲曹雯娟刘志河陈宾汤文彬李孝建
暨南大学附属广州市红十字会医院烧伤整形科
 
Author(s):
JIA Kang ZHANG Zhi LIU Changling CAO Wenjuan LIU Zhihe CHEN Bin TANG Wenbin LI Xiaojian
Department of Burns and Orthopaedics, Guangzhou Red Cross Hospital, Jinan University, Guangzhou, Guangdong Province, 510220, China
 
关键词:
巨噬细胞极化神经调节素受体降解蛋白1转化生长因子&beta1Ⅰ型前胶原
Keywords:
macrophage polarization neuregulin receptor degradation protein 1 transforming growth factor &beta1 procollagen type Ⅰ
分类号:
R341; R363.21; R619.6
文献标志码:
A
摘要:

目的探讨E3连接酶神经调节素受体降解蛋白1(neuregulin receptor degradation protein 1,Nrdp1)调控巨噬细胞极化对成纤维细胞功能的影响。方法分别以内毒素(lipopolysaccharide,LPS)和γ干扰素(interferon-γ,IFN-γ)混合剂、白细胞介素13(interleukin-13,IL-13)诱导小鼠原代腹腔巨噬细胞为M1型巨噬细胞(简称M1)和M2型巨噬细胞(简称M2);流式细胞仪、Western blot分别检测M1表面标记蛋白CD11c、特异性分泌物——诱导型一氧化氮合酶(induciblenitricoxide synthase,iNOS)及M2表面标记蛋白CD206、特异性分泌物——精氨酸酶1(arginine1,Arg1)的表达。转染siRNA-Nrdp1 24 h,Western blot检测Nrdp1表达;建立巨噬细胞与成纤维细胞的Transwell共培养体系,按添加的诱导剂不同,分为LPS组(LPS及IFN-γ共同诱导)、LPS-NC组(siRNA阴性Nrdp1无关序列对照组)、LPS-siRNA-Nrdp1组(转染siRNA-Nrdp1质粒)及对应的IL-13组、IL-13-NC组、IL-13-siRNA-Nrdp1组;培养8、12、24 h,CCK-8检测成纤维细胞增殖情况,ELISA检测转化生长因子β1(transforming growth factor-beta1, TGF-β1)、Ⅰ型前胶原含量。对数据行析因设计方差分析、Bonferroni法检验。结果LPS诱导的巨噬细胞中,M1表面标记蛋白CD11c阳性细胞比例显著高于对照组(P<0.05);IL-13诱导的巨噬细胞中,M2表面标记蛋白CD206阳性细胞比例显著高于未诱导组(P<0.05);LPS组M1特异性分泌物iNOS表达量显著高于对照组(P<0.01)及IL-13组(P<0.01);IL-13组M2型特异性分泌物Arg1显著高于对照组及LPS组(P<0.01)。siRNA-Nrdp1干扰巨噬细胞24 h后,Western blot检测结果显示,干扰组(siRNA-Nrdp1组)Nrdp1的蛋白表达量明显低于空白对照组(P<0.01)及阴性对照组(P<0.01)。共培养24 h后,CCK-8检测结果显示,IL-13-siRNA-Nrdp1组成纤维细胞增殖数量明显低于IL-13组及IL-13-NC组(P<0.01);ELISA检测结果显示,IL-13-siRNA-Nrdp1组TGF-β1含量明显低于IL-13组及IL-13-NC组(P<0.01),IL-13-siRNA-Nrdp1组Ⅰ型前胶原含量明显低于IL-13组(P<0.01)。结论通过siRNA干扰Nrdp1合成,影响巨噬细胞向M2型极化,可抑制M2型巨噬细胞与成纤维细胞共培养体系中成纤维细胞的增殖,减少共培养体系中成纤维细胞TGF-β1及Ⅰ型前胶原的分泌。

Abstract:

ObjectiveTo explore the effect of E3 ligase neuregulin receptor degradation protein 1 (Nrdp1) regulating macrophage polarization on the function of fibroblasts. MethodsLipopolysaccharide (LPS) and interferon-γ (IFN-γ) were combined to induce mouse primary peritoneal macrophages to M1 macrophages (M1), and IL-13 was used to induce macrophages to M2 macrophages (M2). Then flow cytometry and Western blotting were used to detect M1 surface marker protein CD11c and its specific secretion inducible nitric oxide synthase (iNOS), and M2 surface marker protein CD206 and the secretion arginase 1 (Arg1). After the mouse macrophages was transfected with siRNA-Nrdp1 transiently for 24 h, the expression of Nrdp1 was detected by Western blotting. A Transwell co-culture system was established for macrophages and fibroblasts, and was induced under different conditions. The co-cultured cells were divided into LPS group (co-induction of LPS and IFN-γ), LPS-NC group (co-induction and siRNA of irrelevant sequence), LPS-siRNA-Nrdp1 group (co-induction of LPS and siRNA-Nrdp1), IL-13 group, IL-13-NC group and IL-13-siRNA-Nrdp1 group. After co-culture for 8, 12 and 24 h, CCK-8 assay was employed to detect the proliferation of fibroblasts, and ELISA to measure the contents of transforming growth factor β1 (TGF-β1) and type Ⅰ procollagen. Analysis of variance of factorial designs and Bonferroni test were used for statistical analysis for the data. ResultsThe proportion of CD11c positive cells was significantly higher in the LPS-induced macrophages than the non-induced cells (P<0.05), and similar result was seen in the IL-13-induced macrophages for CD206 expression (P<0.05). Western blot analysis indicated the levels of iNOS and Arg1 were obviously higher in the LPS- and IL-13-induced macrophages than the non-induced control cells, respectively (P<0.01). Transient transfection of siRNA-Nrdp1 resulted in notably lower expression of Nrdp1 when compared with the blank control and negative control transfection (both P<0.01). In the co-culture system for 24 h, CCK8 assay showed the count of fibroblasts was significantly lower in the IL-13-siRNA-Nrdp1 group than the IL-13 and IL-13-NC groups (P<0.01), and ELISA indicated that the content of TGF-β1 was lower in the IL-13-siRNA-Nrdp1 group than the IL-13 and IL-13-NC groups (P<0.01), and that of type Ⅰ procollagen was lower in the IL-13-siRNA-Nrdp1 group than the IL-13 groups (P<0.01). ConclusionInterference of Nrdp1 synthesis by siRNA can affect the polarization of macrophages to M2 type, inhibit the proliferation of fibroblasts in the co-culture system of M2 type macrophages and fibroblasts, and reduce the TGF-β1 and type Ⅰ procollagen in the co-culture system.

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更新日期/Last Update: 2021-06-02