Development of anti-cytokine drugs for treatment of endometriosis

开发治疗子宫内膜异位症的抗细胞因子药物

基本信息

批准号：
13671715
负责人：
HARADA Tasuku
金额：
$ 2.56万
依托单位：
Tottori University School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2003
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13671715/
关键词：
endometriosis cytokine interleukin-8 tumor necrosis factor alpha anti-TNF alpha antibody マウスモデル tumor necrosis factor α 抗サイトカイン薬

项目摘要

Peritoneal fluid (PF) in women with endometriosis contains an increased number of activated macrophages that secrete a variety of cytokines. We have previously shown that the PF levels of tumor necrosis factor alpha (TNFa) and interleukin-8 (IL-8) were significantly higher in patients with endometriosis. There was a significant correlation between PF levels of TNFa and IL-8. The purpose of the study is to investigate the role of TNFa in proliferation of endometriotic stromal cells. We also examined the activation of nulear factor-kB (NF-kB) during the induction of IL-8 by TNFa.Chocolate cyst linings of the ovaries in patients with endometriosis (n=39) were a source of endometriotic tissue. The receptors for IL-8 and TNFa were examined by RT-PCR. Proliferation of endometriotic cells was studied using MTT assay. The expression of IL-8 gene and protein was analyzed by Northern blotting and ELISA, respectively. Western blot analysis and electrophoretic mobility shift assay (EMSA) were used … More to detect NF-kB activation by TNFa. Effects of inhibitor for NF-kB, TPCK, on TNFa action were also examined.Transcripts of IL-8 receptor type A, TNF receptor type I and II were detected in endometriotic stromal cells. TNFa (10 and 100 pg/mL) induced the gene and protein expression of IL-8 in endometriotic stromal cells in a dose-dependent fashion. Addition of IL-8 (25-100 pg/mL) or TNFa (5-100 pg/mL) to the culture medium stimulated the proliferation of stromal cells. The stimulatory effects of TNFa were abolished by adding anti-IL-8 antibody. These results suggest that the action of IL-8 mediates the stimulatory effects of TNFa on stromal cell proliferation. The activation of NF-kB is usually associated with phosphorylation of IkB, followed by its degradation by the proteasome and NF-kB nuclear translocation. In order to detect the activation of NF-kB in stromal cells during inducible expression of IL-8 by TNFa, Western blotting was performed using an antibody for phosphorylated IkB (p-IkB). Addition of TNFa (100 pg/mL) rapidly induced p-IkB in stromal cells. EMSA revealed that adding TNFa promoted translocation of activated NF-kB.Preincubation with TPCK reduced TNFa inducible expression of IL-8 gene and protein. TNFa action mediated by IL-8 may contribute to progression of endometriosis by promoting the growth of endometriotic cells. We demonstrated here that NF-kB activation was involved in the induction of IL-8 by TNFa in endometriotic tissues. Thus, TNFa and its signal transduction molecule, NF-kB, play important roles in the pathophysiology of endometriosis. Less

子宫内膜异位症女性的腹膜液（PF）含有增加多种细胞因子的活化巨噬细胞的数量。我们先前已经表明，子宫内膜异位症患者的PF水平肿瘤坏死因子α（TNFA）和白介素8（IL-8）的水平明显更高。 TNFA和IL-8的PF水平之间存在显着相关性。该研究的目的是研究TNFA在子宫内膜基质细胞增殖中的作用。我们还检查了子宫内膜异位症患者的卵巢链球囊肿诱导IL-8期间Nulear Factor-kb（NF-KB）的激活（N = 39）是子宫内膜异位组织的来源。通过RT-PCR检查了IL-8和TNFA的受体。使用MTT分析研究了子宫内膜细胞的增殖。通过Northern印迹和ELISA分析IL-8基因和蛋白质的表达。使用了Western印迹分析和电泳迁移率转移测定法（EMSA）……更多用于检测TNFA的NF-KB激活。还检查了抑制剂对NF-KB，TPCK对TNFA作用的影响。在子宫内膜质细胞中检测到IL-8受体A型，TNF受体I型和II型IL-8受体的转录。 TNFA（10和100 pg/mL）以剂量依赖性的方式诱导子宫内膜含量基质细胞中IL-8的基因和蛋白质表达。在培养基中添加IL-8（25-100 pg/ml）或TNFA（5-100 pg/ml）刺激了基质细胞的增殖。通过添加抗IL-8抗体消除了TNFA的刺激作用。这些结果表明，IL-8的作用介导了TNFA对基质细胞增殖的刺激作用。 NF-KB的激活通常与IKB的磷酸化有关，然后由蛋白酶体和NF-KB核转运降解。为了检测TNFA诱导型IL-8表达期间NF-KB在基质细胞中的激活，使用磷酸化IKB（P-IKB）的抗体进行蛋白质印迹。添加TNFA（100 pg/ml）在基质细胞中迅速诱导的P-IKB。 EMSA透露，添加TNFA促进了活化的NF-KB的转运。与TPCK抗原可降低IL-8基因和蛋白质的TNFA诱导表达。 IL-8介导的TNFA作用可能通过促进子宫内膜细胞的生长来促进子宫内膜异位症的发展。我们在这里证明了NF-KB激活与子宫内膜组织中TNFA诱导IL-8有关。 TNFA及其信号转移分子NF-KB在子宫内膜异位症的病理生理中起重要作用。较少的