THP-1 源巨噬细胞上清液对类风湿性关节炎 成纤维细胞和血管内皮细胞的作用
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科技部国家重点研发计划项目(2018YFC1705205);中国科学院国际合作局国际伙伴计划项目(172644KYSB20190032);国家自然科 学基金委员会面上项目(81672224, 81473709);深圳市科技计划项目基础研究项目(JCYJ20170818153602439, JCYJ20180302150101316);深圳市 医疗卫生三名工程项目(SZSM201808072);深圳市发展和改革委员会工程研究中心扶持计划项目(〔2019〕No. 561.);深圳市工业和信息化局 “创新链+产业链”融合专项项目(201908141541)


The Effect of Supernatant-Derived from THP-1 Macrophage on Rheumatoid Arthritis Fibroblasts and Vascular Endothelial Cells in Vitro
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    摘要:

    在类风湿关节炎(Rheumatoid Arthritis,RA)病理过程中,成纤维细胞的“肿瘤”样转型被认 为与 M1 型巨噬细胞分泌的诱导因子有关,表现为增殖过度、凋亡减少以及异常分泌促炎症因子。此 外,成纤维细胞的异常转型也会通过反馈调节进一步刺激巨噬细胞向 M1 型极化。目前关于 M1 型巨 噬细胞对成纤维细胞和血管内皮细胞的体外作用尚未有报道。为探讨 RA 滑膜中不同细胞间的相互作 用,该研究利用实时荧光定量法检测人髓系白血病单核细胞(THP-1)源 M0、M1 型巨噬细胞中白细 胞介素-8(IL-8)和内皮型一氧化氮合酶(eNOS)基因的 mRNA 表达量,酶联免疫吸附法测定 THP-1 源 M1 型巨噬细胞上清液炎症因子的含量,通过细胞增殖实验测定不同比例 M1 型巨噬细胞上清液对成 纤维细胞和血管内皮细胞的增殖影响。结果显示,THP-1 源 M1 型巨噬细胞主要分泌白细胞介素-1β、 白细胞介素-8 蛋白。对类风湿关节炎滑膜成纤维细胞(MH7A)而言, M1 型巨噬细胞上清液与含血 清 RPMI(Roswell Park Memorial Institute)培养基混合(比例为 7∶3)作用 48 h 能促进细胞增殖;对人脐 静脉细胞融合细胞(EA.hy926)而言,M1 型巨噬细胞上清液与含血清 DMEM(Dulbecco Modified Eagle Medium)培养基混合(比例为 6∶4 或 7∶3)处理 72 h 显著促进了细胞的增殖。该研究可为建立 RA 血管 翳类组织模型提供前期研究的数据支撑,有助于体外构建抗 RA 药物高通量筛选平台。

    Abstract:

    Normal synovium is mainly composed of fibroblasts and macrophages. In the pathological process of rheumatoid arthritis (RA), the “tumor like” transformation of fibroblasts is considered to be related to inducers secreted by M1 macrophages. They are manifested as excessive proliferation, decreased apoptosis and abnormal secretion of pro-inflammatory factors, which in turn stimulate the macrophages for M1 polarization. However, the effect of M1 macrophages on fibroblasts and vascular endothelial cells has not been reported. In this study, we aim to reveal the interactions between different types of cells in the RA synovium. mRNA expression of IL-8 and eNOS in M0 and M1 macrophages derived from human myeloid leukemia monocytes (THP-1) were detected by real-time fluorescence quantitative method. Moreover, the concentration of inflammatory factors in the supernatant of THP-1 derived M1 macrophages was determined by enzymelinked immunosorbent assay. In addition, the proliferation of fibroblasts and vascular endothelial cells was investigated by CCK8. The results showed that interlekin-1β and interlekin-8 proteins were mainly secreted by THP-1 derived M1 macrophages. We found that the rheumatoid arthritis synovial fibroblasts (MH7A) treated with mixture of M1 macrophage supernatant and serum-containing roswell park memorial institute (RPMI) with the ratio of 7∶3 for 48 h, and the cells proliferation was improved. Meanwhile, the proliferation of human umbilical vein cells was remarkable enhanced after treated with the mixture ratio at 6∶4 or 7∶3 for 72 h. Based on this study, we will further establish the in vitro RA pannus model in future, which will facilitate the generation of high-throughput screening techniques for anti-RA drugs development.

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引文格式
林结桃,李 健,成文翔,等. THP-1 源巨噬细胞上清液对类风湿性关节炎 成纤维细胞和血管内皮细胞的作用 [J].集成技术,2020,9(4):1-10

Citing format
LIN Jietao, LI Jian, CHENG Wenxiang, et al. The Effect of Supernatant-Derived from THP-1 Macrophage on Rheumatoid Arthritis Fibroblasts and Vascular Endothelial Cells in Vitro[J]. Journal of Integration Technology,2020,9(4):1-10

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  • 在线发布日期: 2020-07-21
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