BIOLOGY OF TRABECULAR MESHWORK IN HEALTH AND DISEASE

小梁网在健康和疾病中的生物学

• 批准号: 2159497
• 负责人: BEATRICE Y.J.T. YUE
• 金额: $ 16.62万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-12-01 至 1995-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2159497
• 关键词: affinity chromatography; aging; cell adhesion; cell motility; collagen; dexamethasone; elastases; enzyme linked immunosorbent assay; extracellular matrix proteins; fibronectins; gene expression; glaucoma; glucocorticoids; human tissue; immunologic techniques; in situ hybridization; integrins; intraocular pressure; laminin; northern blottings; organ culture; protease inhibitor; protein biosynthesis; tissue /cell culture; trabecular meshwork

The trabecular meshwork is believed to be a major site for the regulation of the aqueous outflow. Residing in this tissue are trabecular meshwork cells that are essential for the maintenance of normal outflow. Aberrations of cell integrity may be a key step toward obstruction of the aqueous outflow, intraocular pressure elevations, and glaucomatous conditions. Besides the cells, extracellular matrix (ECM) materials such as fibronectin, laminin, and collagens are also thought to be important for normal functioning of the trabecular meshwork. We propose, in this application, to examine the human trabecular meshwork for the integrins, a family of receptors for ECM elements and other cell-surface proteins, that can mediate cell adhesion, influence cellular activities, and affect the growth, morphology, metabolism, and differentiation of cells. Specifically, the types and quantity of the receptors for fibronectin, laminin, and collagens in human trabecular meshwork tissues and in primary, first- and second-passage cell cultures will be determined by gel electrophoresis, affinity chromatography, immunoprecipitation, and other methods. Since primary open angle glaucoma (POAG) is known to be an age- related disorder, specimens obtained from donors of three different age groups (< 20, 21-49, and > 50 years) will be studied to determine whether variations exist with aging. Roles of integrins in attachment and spreading of cultured trabecular meshwork cells will also be evaluated. We will further assess the composition of collagens in human trabecular meshwork tissues and cell cultures. We have shown that collagen types I, III, IV, V, and VI are expressed in the human trabecular meshwork. Our investigations will be expanded to include studies for collagen type VIII and to systematically and quantitatively evaluate the age-related changes in the gene expression of collagens. Protocols for Northern blot, in situ hybridization, enzyme-linked immunosorbent assay (ELISA), and biochemical analyses will be used. In addition, the effects of glucocorticoids on trabecular meshwork cells in both cell culture and organ culture systems will be evaluated. Glucocorticoids such as dexamethasone induced intraocular pressure elevations in susceptible humans and rabbits, and the underlying alterations in the trabecular meshwork have bene suggested to be similar to those seen in POAG. In our study, the modulation by dexamethasone in the production of fibronectin, laminin, collagens, and integrins will be assessed by techniques including immunostaining, ELISA, and Western blotting. Variations in mRNA levels will be documented by molecular biology methods. Moreover, changes induced by dexamethasone in levels of cathepsins B and G, elastase, and alpha1-proteinase inhibitor, all of which are pivotal for the turnover and remodeling of the ECM, will be investigated by immunohistochemical staining, biochemical assays, and ELISA. mRNA levels for cathepsin G and alpha1-proteinase inhibitor will also be measured to delineate the effects of dexamethasone. Our goal is to further elucidate the biologic characteristics of healthy as well as experimentally-altered trabecular meshwork cells, particularly with regard to the dynamics and control mechanisms involved in the synthesis and turnover of ECM proteins in the trabecular meshwork. This information will assist in uncovering the pathogenic processes of POAG.

据信小梁网络被认为是该法规的主要地点 水流流出。 居住在该组织中的是小梁网 对于维持正常流出必不可少的单元格。 细胞完整性的畸变可能是障碍的关键步骤 水流出，眼内压力和青光眼 状况。 除细胞外，细胞外基质（ECM）材料此类 由于纤连蛋白，层粘连蛋白和胶原蛋白也被认为对 小梁网的正常功能。 我们提出了这一点 应用，检查整联蛋白的人小梁网络 ECM元素和其他细胞表面蛋白的受体家族， 可以介导细胞粘附，影响细胞活性，并影响 细胞的生长，形态，代谢和分化。 具体而言，纤连蛋白的受体的类型和数量， 层粘连蛋白和人的小梁网络组织中的胶原蛋白和原发性的胶原蛋白 第一和第二-Passage细胞培养物将由凝胶确定 电泳，亲和色谱，免疫沉淀和其他 方法。 由于原发性开角青光眼（POAG）已知是年龄的 相关障碍，从三个不同年龄的捐助者获得的标本 将研究组（<20，21-49和> 50年），以确定是否 随着衰老而存在变化。 整合素在附件中的作用和 还将评估培养的小梁网细胞的扩散。 我们将进一步评估人小梁中胶原蛋白的组成 网状组织和细胞培养物。 我们已经证明了胶原蛋白I类型， III，IV，V和VI在人的小梁网中表达。 我们的 调查将扩大到包括胶原蛋白型的研究 并进行系统和定量评估与年龄有关的变化 在胶原蛋白的基因表达中。 北印迹的协议，原位 杂交，酶联免疫吸附测定（ELISA）和生化 将使用分析。 另外，糖皮质激素对小梁网细胞的影响 将评估细胞培养和器官培养系统。 糖皮质激素（例如地塞米松）诱导的眼压 易感人类和兔子的海拔 小梁网的改变已提出与 那些在Poag中看到的。 在我们的研究中，地塞米松在 纤连蛋白，层粘连蛋白，胶原蛋白和整联蛋白的产生将是 通过包括免疫染色，ELISA和西方在内的技术评估 吸毒。 mRNA水平的变化将通过分子记录 生物学方法。 此外，地塞米松在水平上引起的变化 组织蛋白酶B和G，弹性酶和α1-蛋白酶抑制剂，所有这些 对于ECM的营业额和重塑是关键的，将是 通过免疫组织化学染色，生化测定和 Elisa。 组织蛋白酶G和α1-蛋白酶抑制剂的mRNA水平将 还可以测量以描绘地塞米松的影响。 我们的目标是 进一步阐明健康的生物学特征 实验室改变的小梁网络工作单元，尤其是关于 涉及合成和控制机制 小梁网中ECM蛋白的营业额。 这些信息将 协助发现POAG的致病过程。