【作者】 刘娅利；

【导师】 陈晓明； 刘旭阳；

【作者基本信息】 四川大学 ， 眼科学， 2007， 博士

【摘要】 目的：研究糖皮质激素(地塞米松)和Rho激酶抑制剂Y-27632对体外培养的人小梁细胞及细胞骨架的影响。采用局部滴用糖皮质激素联合结膜下注射曲安耐德建立糖皮质激素性猫高眼压模型。观察Y-27632对猫糖皮质激素性高眼压的降眼压效果。探讨激素性青光眼的发病机制以及Y-27632的降眼压机制。方法：1．采用第6代人小梁细胞，培养至细胞融合为稳定的内皮细胞样形态，加入地塞米松(200mM)继续培养2周，然后用1 0gM Y-27632分别处理细胞3小时和24小时，观察细胞形态以及肌动蛋白及粘着斑蛋白的变化。2．成年家猫被训练接受局部滴眼药，并适应在表面麻醉下进行眼压测量。采用TONO-PEN XL眼压计，连续3天每天测量双眼眼压，取平均值作为基础眼压。然后采用0.5％地塞米松滴右眼，3次／天，局部麻醉下结膜下注射3mg曲安耐德，每周1次。左眼仅滴生理盐水作为对照。使用糖皮质激素后每周测量1次眼压，并观察眼的局部反应和全身反应。8周后，采集猫小梁网标本送电镜检查。3．将眼压升高的猫纳入进一步降眼压研究，取高眼压眼分成3组分别滴30mM，10mM浓度的Y-27632和对照液PBS。用微量加样器将Y-27632或PBS点到角膜中央，每次5ul，间隔30see，共点3次。用TONO-PEN XL眼压计测量滴眼后0.5、1、3、6、9、24小时眼压。结果：1．与正常培养的人小梁细胞比较，地塞米松处理的小梁细胞表现肌动蛋白增加，形成肌动蛋白交联网，同时粘着斑蛋白表达增多。用Y-27632处理后，小梁细胞回缩变圆，肌动蛋白降解或减少，粘着斑明显减少或趋于消失。中止Y-27632作用后，肌动蛋白及粘着斑蛋白恢复，表明Y-27632的作用有可逆性。2．32只实验猫的平均基础眼压为18.64±2.42 mmHg。在接受糖皮质激素的28眼中，25眼在用药第2周眼压开始升高，到第6周时达到峰值。然后保持一个相对高平台。从第2周开始，实验组眼压升高峰值与同期对照组眼压比较，平均差值达7.38mm Hg (P＜0.05)。在激素诱导的高眼压眼，电镜检查，小梁细胞核异形性增大，细胞间质内可见微绒毛样结构，粗面内质网扩张，细胞间基质增多。3．Y-27632对猫糖皮质激素性高眼压有明显的降眼压效果。与空白对照组比较，Y-27632作用于激素诱导的猫高眼压眼半小时后眼压即开始显著下降(P＜0.05)。用药1小时后眼压下降到最低点。浓度为30mM的Y-27632降眼压效果在用药后0.5～3小时之间明显，眼压平均最大下降幅度为6.13mmHg，浓度为10mM的Y-27632降眼压效果在用药后0.5～1小时之间明显，平均最大下降幅度为5.12mmHg。结论：1．在地塞米松作用下，人小梁细胞肌动蛋白细胞骨架形成肌动蛋白交联网结构，粘着斑蛋白表达增多。推测糖皮质激素诱导小梁细胞骨架改变是激素性青光眼眼压升高的原因。Rho激酶抑制剂Y-27632可有效降解地塞米松所引起的小梁细胞骨架改变，提示Y-27632可能降低糖皮质激素性高眼压。2．家猫对眼局部应用糖皮质激素反应率高，89％实验眼发生了糖皮质激素诱导的高眼压。糖皮质激素诱导的猫高眼压模型对了解糖皮质激素性青光眼的发病机制具有重要意义，也可用于寻找和筛选有效的降眼压药物。3．Rho激酶抑制剂Y-27632能降低猫糖皮质激素性高眼压，降眼压的持续时间和降压幅度呈剂量依赖性。Rho激酶抑制剂Y-27632是一种有潜力的抗青光眼药物，其降眼压机制可能是通过改变小梁细胞骨架发挥作用。更多还原

【Abstract】 PURPOSE: To investigate the roles of glucocorticoid (Dexamethasone, DEX) and Rho kinase inhibitor. Y-27632 the cytoskeleton on human trabecular meshwork (HTM) cells in vitro. To establish a kind model of corticosteroid -induced elevation of IOP in cats by topical adminstration of Dexamethasone drops and subconjunctival injections of triamcinoIone.To observe the role of Rho kinase inhibitor Y-27632 in the regulating intraocular pressure in the cats with corticosteroid-induced ocular hypertension. To investigate the pathogenesis of corticosteroid-induced glaucoma and the mechanism lowering the intraocular pressue of Rho kinase inhibitor Y-27632.METHODS:1. HTM cells (sixth passage) were cultured to a confluent stage with stableendothelium-like morphology and incubated with DEX (200mM) for 2 weeks. Then the cells were treated with 10μM of Y-27632 for 3, 24hrs respectively. Then we observed the changs on cell morphology and actin and vinculin.2. Adult domestic cats were trained to accept ocular drug administration andtonometry under topical anesthesia. To establish intraocular pressure (IOP) baseline IOP values, tonometry was performed at the same time of day for 3 consecutive days, with TONO-PEN XL tonometer. Beginning on day 4, thirty-two cats received either 0.5% dexamethasone (right eye) or vehicle (left eye) administered topically three times a day for approximately 56 days, and subconjunctival injections of 3mg triamcinolone in right eyes under topical anesthesia weekly. IOP was monitored once a week after the corticosteroid administration. Then, the tissue of trabecular meshwork (TM) was collected and was examined by electric microscopy.3. The cats with corticosteroid-induced ocular hypertension were used in this study. Rho kinase-specific inhibitor, Y-27632, was topically administered to one eye of the cats with corticosteroid-induced ocular hypertension.Y-27632 (30mM or 10mM) or phosphate buffered saline (PBS) was administered to the central cornea as three 5-μl drops at intervals of 30 seconds. TONO-PEN XL tonometer was used to monitor the IOP. It was measured before the administration of Y-27632 and at 0.5, 1, 3, 6, 9, and 24 hours after administration.RESULTS:1. Compared with DEX-free cultured HTM cells, there was an increased amount of the actin cytoskeleton, even formed cross-linked actin networks, and vinculin-positive focal adhesions increased in DEX-treated cells. Y-27632 treated HTM cells appeared to be retracted and round up. The actin cytoskeleton was disrupted or reduced, vinculin-positive focal adhesions were reduced in number or disappeared in Y-27632 treated cells. These alterations were found to be reversible after drug was withdrawn. 2. The baseline IOP of the rest 32 cats was 18.64±2.42 mmHg. Introcular pressure began to increase after 2 weeks of treatment in 89% of the cat eyes receiving corticosteroid, and reached a peak 4 weeks later. The peak IOP differences between the corticosteroid-treated eye and the fellow control eye reached up to 7.38mmHg (P<0.05). The changes of the trabecular cells ultrastucture, in the corticosteroid-induced hypertension cat eyes, including dysmorphic change of nucleus, enlargement of the endoplasmic and Golgi apparatus, and deposition of TM extracellular matrix.3. In cat eyes with corticosteroid-induced ocular hypertension, topical administration of Y-27632 resulted in a significant decrease in IOP. Compared with PBS-treated control eyes, the IOP in Y-27632-treated eyes was significantly lowered 0.5 hours after topical administration of Y-27632 eye drops (P < 0.05). The average maximum decreases was seen after 60 minutes administration of Y-27632 .The IOP reduction was observed between 0.5 and 3 hours (P < 0.05) with 30 mM of Y-27632 and the average maximum decreases was 6.13mmHg. With the 10mM concentration eye drops, the reduction were seen between 0.5 and 1 hour (P < 0.05) and the average maximum decreases was 5.12mmHg.CONCLUSIONS:1. Corticosteriod caused an obvious change in the organization of actin cytoskeleton and vinculin-positive focal adhesions in the cultured HTM cells.we infer that the cocorticoid-mediated changes in the HTM cytoskeleton maybe the pathogenesis of corticosteroid-induced glaucoma. Because Y-27632 can inhibit the DEX-induced changes in the HTM cells. It indicate that Rho kinase inhibitor Y-27632 can decrease the cocorticoid-induced intraocular pressure.2. The domestic cat eyes exhibit a robust steroid-induced ocular hypertensive response, with 89% occurrence in this trial. The high prevalence of corticosteroid-induced elevation of IOP in the cat eyes will permit studies on the mechanism of steroid-induced glaucoma and the pressure-lowering effect of drugs.3. Administration of Y-27632 caused a reduction in IOP in cat eyes with corticosteroid-induced ocular hypertension in a dose-dependent manner. The Rho kinase-specific inhibitor Y-27632 is a promising treatment for glaucoma therapy in the next generation and IOP-lowering effects of Y-27632 may be related to the altered cytoskeleton behavior of TM cells.更多还原