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

• 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

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

子宫内膜异位症患者的腹腔液（PF）中含有大量活化的巨噬细胞，这些巨噬细胞分泌多种细胞因子。我们先前已经表明，肿瘤坏死因子α（TNF α）和白细胞介素-8（IL-8）的PF水平在子宫内膜异位症患者中显著升高。TNF α和IL-8的PF水平之间存在显著相关性。本研究的目的是探讨肿瘤坏死因子α（TNF α）在肿瘤间质细胞增殖中的作用。我们还检测了TNF α诱导IL-8过程中核因子-kB（NF-kB）的激活。子宫内膜异位症患者（n=39）卵巢巧克力囊肿衬里是异位组织的来源。RT-PCR检测IL-8和TNF α受体。MTT法检测细胞增殖情况;分别采用北方印迹法和ELISA法检测IL-8基因和蛋白的表达。采用蛋白质印迹分析和电泳迁移率变动分析（EMSA） ...更多信息 检测TNFa对NF-kB的激活。检测了TNF α对NF-κ B抑制剂TPCK的作用，并检测了炎症基质细胞中IL-8受体A型、TNF受体I型和II型的转录。TNF α（10和100 pg/mL）以剂量依赖性方式诱导增生基质细胞中IL-8的基因和蛋白表达。向培养基中添加IL-8（25-100 pg/mL）或TNFa（5-100 pg/mL）刺激基质细胞的增殖。加入抗IL-8抗体可消除TNF α的刺激作用。这些结果表明IL-8的作用介导TNF α对基质细胞增殖的刺激作用。NF-κ B的活化通常与IkB的磷酸化，随后通过蛋白酶体和NF-κ B核转位降解有关。为了检测在TNF α诱导表达IL-8期间基质细胞中NF-κ B的活化，使用磷酸化IkB（p-IkB）的抗体进行蛋白质印迹。添加TNF α（100 pg/mL）快速诱导基质细胞中的p-IkB。EMSA结果显示，TNF α可促进活化的NF-κ B转位，TPCK预孵育可降低TNF α诱导的IL-8基因和蛋白表达。IL-8介导的TNF α作用可能通过促进子宫内膜异位症细胞的生长而促进子宫内膜异位症的进展。我们在此证明，NF-κ B活化参与了肿瘤组织中TNF α诱导IL-8的过程。因此，TNFa及其信号转导分子NF-kB在子宫内膜异位症的病理生理学中起重要作用。少

项目成果

A.Fujii: "Interleukin-8 expression is up-regulated by interleukin-1β in steroidogenic human granulose-like cells."Fertil Steril. 79. 151-157 (2003)

A.Fujii：“在类固醇生成人颗粒样细胞中，白细胞介素 8 的表达被白细胞介素 1β 上调。”Fertil Steril，79. 151-157 (2003)

S.Yoshida: "Laparoscopic surgery for the management of ovarian endometrioma."Gynecol Obstet Invest. 54. 24-29 (2002)

S.Yoshida：“腹腔镜手术治疗卵巢子宫内膜异位症。”Gynecol Obstet Invest。

A.Fujii: "Interleukin-8 gene and protein expression are up-regulated by interleukin-1β in normal human ovarian cells and a granulosa tumor cell line"Fertil Steril. 79(2). 151-157 (2003)

A.Fujii：“正常人卵巢细胞和颗粒肿瘤细胞系中白细胞介素 1β 上调白细胞介素 8 基因和蛋白质表达”Fertil Steril 79(2) (2003)。

S.Yoshida: "Hepatocyte growth factor (HGF)/Met system promotes endometrial and endometriotic stromal cell invasion via autocrine and paracrine pathway."J Clin Endocrinol Metab. 89. 823-832 (2004)

S.Yoshida：“肝细胞生长因子 (HGF)/Met 系统通过自分泌和旁分泌途径促进子宫内膜和子宫内膜异位基质细胞侵袭。”J Clin Endocrinol Metab。

T.Harada: "The role of cytokines in endometriosis."Fertil Steril. 6(1). 1-10 (2001)

T.Harada：“细胞因子在子宫内膜异位症中的作用。”Fertil Steril。