UROTHELIAL CELL ACTIVATION IN INTERSTITIAL CYSTITIS

间质性膀胱炎中的尿路上皮细胞激活

• 批准号: 3246277
• 负责人: Monica Liebert
• 金额: $ 18.15万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-09-30 至 1994-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3246277
• 关键词: MHC class II antigen; biopsy; cell adhesion molecules; cell cell interaction; cyclosporines; cytokine; enzyme linked immunosorbent assay; fibroblasts; flow cytometry; gene expression; human tissue; immunofluorescence technique; immunosuppressive; interferons; interleukin 1; interstitial cystitis; northern blottings; polymerase chain reaction; protein biosynthesis; tissue /cell culture; tumor necrosis factor alpha; urinary bladder

Interstitial cystitis is a poorly understood inflammatory condition occurring in the urinary bladder. We hypothesize that this condition is due to or is exacerbated by the production of cytokines by the urothelial cells of the bladder, and that the local action of immunosuppressive drugs, such as Cyclosporin A, FK506, or uromodulin, will reduce or halt this cellular activation and cytokine production and play a role in controlling the symptoms of IC. To evaluate these hypotheses, we propose these studies: Specific Aim 1: To characterize the cytokine production and expression of an activated phenotype (induced expression of cell surface antigens such as HLA-DR or intercellular adhesion molecule-1 [ICAM-1]) by urothelial cells in vitro. These studies will be performed using human cultured normal urothelial cells. These urothelial cells will be cultured in vitro with stimulants such as gamma interferon, tumor necrosis factor alpha, and interleukin 1. When activated, these cells will respond by producing cytokines and/or expressing cell surface antigens such as HLA-DR or ICAM-1. The evaluation of cell surface antigen expression will be performed by enzyme-linked immunosorbent assay (ELISA) and/or flow cytometry. The production of cytokines will be evaluated by the messenger RNA (mRNA) polymerase chain reaction (PCR) and by Northern blotting. PCR, will be used because it will allow the evaluation of multiple cytokines from very small samples. Bioactivity of the cytokines produced will be evaluated by bioassay. Interactions between urothelial cells and fibroblasts will be performed in a two-phase culture model. Either or both cell types will be stimulated and both cell types will be evaluated for cell surface antigen phenotype and cytokine production and for proliferation or growth inhibition using a dye-binding technique. Specific Aim 2: To characterize the cytokine production and expression of an activated phenotype in vivo. Tissue specimens taken from patients with interstitial cystitis and normal donors will be evaluated for cytokine production and cell surface antigen expression. These specimens will be analyzed for antigen expression by immunofluorescence staining and for cytokine production by RNA PCR analysis. Specific Aim 3: To determine if immunosuppressant drugs can affect cytokine generation and other markers of cellular activation in vitro. These studies will be performed using in vitro stimulated cultured normal urothelial cells treated with drug. The cytokine response in the presence or absence of drug (such as Cyclosporin A, FK506, or uromodulin) will be evaluated by RNA PCR or Northern blotting. Co-cultures of fibroblasts and normal urothelial cells will also be evaluated in this model with or without treatment with drug. These studies will not only further our understanding of the biology of urothelial cells and their participation in inflammatory processes, but also may identify new diagnostic criteria for a subset of interstitial cystitis patients and may yield a new method of treatment for this disease.

间质性膀胱炎是一种人们知之甚少的炎症性疾病 发生在膀胱。 我们假设这个条件是 由于尿路上皮产生细胞因子或因尿路上皮产生细胞因子而加剧 膀胱细胞，以及免疫抑制药物的局部作用， 例如环孢素 A、FK506 或尿调节素，会减少或阻止这种情况 细胞活化和细胞因子产生并在控制中发挥作用 IC 的症状。 为了评估这些假设，我们提出这些 研究： 具体目标 1：表征细胞因子的产生和表达 激活的表型（诱导细胞表面抗原的表达，例如 尿路上皮细胞的 HLA-DR 或细胞间粘附分子-1 [ICAM-1]） 体外。 这些研究将使用人类培养的正常细胞进行 尿路上皮细胞。 这些尿路上皮细胞将在体外培养 兴奋剂，例如γ干扰素、肿瘤坏死因子α和 白细胞介素 1。当被激活时，这些细胞将产生反应 细胞因子和/或表达细胞表面抗原，例如 HLA-DR 或 ICAM-1。 细胞表面抗原表达的评估将通过 酶联免疫吸附测定（ELISA）和/或流式细胞术。 这 细胞因子的产生将通过信使 RNA (mRNA) 进行评估 聚合酶链反应 (PCR) 和 Northern 印迹法。 PCR，将 使用它是因为它可以评估多种细胞因子 小样本。 产生的细胞因子的生物活性将通过以下方式进行评估 生物测定。 尿路上皮细胞和成纤维细胞之间的相互作用 在两相培养模型中进行。 一种或两种细胞类型将是 刺激并且将评估两种细胞类型的细胞表面抗原 表型和细胞因子的产生以及增殖或生长 使用染料结合技术进行抑制。 具体目标 2：表征 体内激活表型的细胞因子产生和表达。 取自间质性膀胱炎患者和正常人的组织标本 将评估捐赠者的细胞因子产生和细胞表面抗原 表达。 将通过以下方式分析这些样本的抗原表达： 免疫荧光染色和通过 RNA PCR 产生细胞因子 分析。具体目标 3：确定免疫抑制剂是否可以 影响细胞因子的产生和细胞激活的其他标志物 体外。 这些研究将使用体外刺激培养物进行 用药物处理的正常尿路上皮细胞。 细胞因子反应在 是否存在药物（例如环孢素 A、FK506 或尿调节素） 将通过 RNA PCR 或 Northern blotting 进行评估。 共培养 成纤维细胞和正常尿路上皮细胞也将在此进行评估 有或没有药物治疗的模型。 这些研究不仅将 进一步加深我们对尿路上皮细胞及其生物学特性的理解 参与炎症过程，但也可能发现新的 部分间质性膀胱炎患者的诊断标准，可能 为治疗这种疾病提供了一种新方法。