CELLULAR AND MOLECULAR BIOLOGY OF CHOLESTEATOMA

耳源性中耳瘤起源于鼓膜，是中耳炎的后遗症。 中耳炎 胆脂瘤是一种进行性上皮病变， 破坏中耳的骨质结构，有时甚至是内耳。 一旦确定，中耳瘤只能通过外科手术 根除和二期中耳重建。 长期 主要研究者研究计划的目标是 了解导致发展的细胞机制， 进展和骨质破坏。 如此之低- 站立可能导致非手术预防策略， 管理这种疾病。 本申请被设计为研究以下关系： 两种重要的细胞间信号分子白细胞介素-1（IL-1） 和一氧化氮（NO）对中耳炎性骨侵蚀的影响。 本申请的具体目的是：1）确定 细胞因子的白细胞介素家族（IL-1 α，IL-1 β和IL-1 ra）在实验性中耳炎中的作用， 压力诱导的沙土鼠破骨细胞募集和激活 同步骨建模的大疱模型;和2）研究同种型 一氧化氮合酶（NOS I，II，III）在局部控制 破骨细胞在体外和体内。 有两种广泛的假设，每一种都集中在IL-1的同种型上， 号 对于特异性Aim 1，将检验假设1：IL-1亚型是 转录在骨衬里细胞和成纤维细胞的亚- 位于中耳乳突瘤下的上皮和受压的大泡中， 代表了一个早期的信号，为招聘和激活 局部破骨细胞。 实验性沙鼠乳头瘤， 沙鼠气压泡，PCS，PCR产物克隆，DNA 测序、定量RT-PCR、骨组织形态计量学和RNA原位 杂交将被用来检验第一个假设。 对于特定目标2，将检验假设2：NO是由上-下产生的。 骨衬里细胞和成纤维细胞内NOS亚型的调节 乳突瘤下的亚上皮和加压的 大疱。 这导致了NO的本地化和招聘 和破骨细胞的活化。 小鼠颅骨器官培养 （元素钙释放和45 Ca释放），沙鼠碱加压 大疱，PCR，PCR产物克隆，DNA测序，定量RT-PCR PCR、骨组织形态计量学、RNA原位杂交和NADPH- 黄递酶组织化学将用于检验第二种假设。

Aural cholesteatomas arise from the tympanic membrane as a sequela of otitis media. Cholesteatomas are progressive, epithelial lesions that destroy the bony structures of the middle and, sometimes, inner ear. Once established, cholesteatomas can only be mandated by surgical eradication and secondary middle ear reconstruction. The long-term objective of the Principal Investigator's research program is to understand the cellular mechanisms that lead to the development, progression, and bone destruction of cholesteatomas. Such an under- standing may lead to strategies for the non-surgical prevention and management of this disease. The present application is designed to investigate the relationship of two important intercellular signaling molecules, interleukin-1 (IL-1) and nitric oxide (NO), to osteoclastic bone erosion in cholesteatoma. The specific aims of this application are: 1) to determine the expression of the interleukin family of cytokines (IL-1 alpha, IL-1 beta, and IL-1ra) in experimental cholesteatoma and also during osteoclast recruitment and activation induced by pressure in the gerbil bulla model of synchronous bone modeling; and 2) to investigate isoforms of nitric oxide synthase (NOS I, II, III) in the local control of osteoclasts in vitro and in vivo. There are two broad hypothesis, each focused on the isoforms of IL-1 and NOS. For Specific Aim1, hypothesis 1 will be tested: IL-1 isoforms are transcribed within bone lining cells and fibroblasts of the sub- epithelium subjacent to cholesteatoma and in the pressurized bulla and represent an early signal for the recruitment and activation of osteoclasts at a local site. Experimental gerbilline cholesteatoma, gerbilline pressurized bulla, PCS, cloning of PCR products, DNA sequencing, quantitative RT-PCR, bone histomorphometry, and RNA in situ hybridization will be used to test this first hypothesis. For Specific Aim 2, hypothesis 2 will be tested: NO is produced by up- regulation of NOS isoforms within bone lining cells and fibroblasts of the sub-epithelium subjacent to cholesteatoma and in the pressurized bulla. This leads to the local elaboration of NO and the recruitment and activation of osteoclasts. Mouse calvarial organ culture (elemental calcium release and 45Ca release), gerbilline pressurized bulla, PCR, cloning of PCR products, DNA sequencing, quantitative RT- PCR, bone histomorphometry, RNA in situ hybridization, and NADPH- diaphorase histochemistry will be used to test this second hypothesis.