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: 2144051
• 负责人: Monica Liebert
• 金额: $ 19.09万
• 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 1995-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2144051
• 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; interferon gamma; 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知之甚少的炎症性疾病。 发生在膀胱里的。我们假设这种情况是 由于尿路上皮产生的细胞因子或因此而加重的 膀胱细胞，免疫抑制药物的局部作用， 如环孢菌素A、FK506或尿调素，将减少或阻止这种情况 细胞活化和细胞因子的产生及其在调控中的作用 IC的症状。为了评估这些假设，我们提出了以下几点 研究： 特异性目标1：鉴定细胞因子的产生和表达 一种激活的表型(诱导细胞表面抗原的表达，如 细胞间黏附分子-1[ICAM-1]) 在试管中。这些研究将使用人类培养的正常组织进行 尿路上皮细胞。这些尿路上皮细胞将在体外培养 刺激物，如干扰素、肿瘤坏死因子α和 白介素1。当激活时，这些细胞将通过产生 细胞因子和/或表达细胞表面抗原，如人类白细胞抗原DR或细胞间黏附分子-1。 细胞表面抗原表达的评估将通过以下方式进行 酶联免疫吸附试验(ELISA)和/或流式细胞术。这个 细胞因子的产生将通过信使核糖核酸(信使核糖核酸)来评估。 聚合酶链式反应(PCR)和Northern杂交。PCR，将是 使用它，因为它将允许评估多种细胞因子从非常 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 生物化验。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。任一或两种单元格类型都将是 将对刺激的两种细胞类型进行细胞表面抗原的评估 表型和细胞因子的产生以及对增殖或生长的影响 使用染料结合技术的抑制。具体目标2：确定 体内激活表型的细胞因子的产生和表达。 间质性膀胱炎患者和正常人的组织标本 捐赠者将接受细胞因子产生和细胞表面抗原的评估。 表情。这些样本将通过以下方式分析抗原表达 免疫荧光染色和RNA-PCR检测细胞因子的产生 分析。具体目标3：确定免疫抑制药物是否可以 影响细胞因子的产生和其他细胞激活的标志 体外培养。这些研究将使用体外刺激培养的方法进行 药物处理正常的尿路上皮细胞。血管内皮细胞中的细胞因子反应 有无药物(如环孢素A、FK506或尿调素) 将通过RNA聚合酶链式反应或Northern blotting进行鉴定。的共生文化 成纤维细胞和正常的尿路上皮细胞也将在这方面进行评估 接受或不接受药物治疗的模特。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的了解 参与炎症过程，但也可能识别新的 间质性膀胱炎患者亚组的诊断标准 产生了一种治疗这种疾病的新方法。