UROTHELIAL CELL ACTIVATION IN INTERSTITIAL CYSTITIS

间质性膀胱炎是一种知之甚少的炎症性疾病 发生在膀胱中。 我们假设这种情况是 由于尿路上皮细胞产生细胞因子或因其而加重 膀胱的细胞，以及免疫抑制药物的局部作用， 如环孢菌素A、FK506或尿调节素，将减少或停止这种作用 细胞活化和细胞因子的产生，并在控制 IC的症状。 为了评估这些假设，我们提出了以下建议： 研究： 具体目的1：表征细胞因子的产生和表达， 活化表型（诱导细胞表面抗原的表达， HLA-DR或细胞间粘附分子-1（ICAM-1） 体外 这些研究将使用人培养的正常 尿路上皮细胞 这些尿路上皮细胞将在体外培养， 刺激剂如γ干扰素、肿瘤坏死因子α，和 白细胞介素1。当被激活时，这些细胞会产生 细胞因子和/或表达细胞表面抗原如HLA-DR或ICAM-1。 细胞表面抗原表达的评价将通过 酶联免疫吸附测定（ELISA）和/或流式细胞术。 的 细胞因子的产生将通过信使RNA（mRNA） 聚合酶链反应（PCR）和北方印迹法。 PCR，将是 使用，因为它将允许评估多种细胞因子， 小样本。 将通过以下方法评价产生的细胞因子的生物活性： 生物测定 尿路上皮细胞和成纤维细胞之间的相互作用将是 在两相培养模型中进行。 其中一种或两种细胞类型将被 刺激，两种细胞类型将评价细胞表面抗原 表型和细胞因子产生以及增殖或生长 抑制使用染料结合技术。 具体目标2：描述 体内细胞因子的产生和活化表型的表达。 组织标本取自间质性膀胱炎患者和正常人 将评价供体的细胞因子产生和细胞表面抗原 表情 将通过以下方法分析这些标本的抗原表达： 免疫荧光染色和通过RNA PCR用于细胞因子产生 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细胞活化的其他标志物， 体外 这些研究将使用体外刺激培养的 用药物处理的正常尿路上皮细胞。 细胞因子反应 是否存在药物（如环孢菌素A、FK506或尿调节素） 将通过RNA PCR或北方印迹法进行评价。 的共培养物 成纤维细胞和正常的尿路上皮细胞也将在此进行评估。 有或无药物治疗的模型。 这些研究不仅 进一步了解尿路上皮细胞的生物学及其 参与炎症过程，但也可能发现新的 间质性膀胱炎患者亚组的诊断标准， 为这种疾病提供了一种新的治疗方法。

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.