盐酸法舒地尔对离体牛眼房水流畅系数及牛眼小梁细胞骨架的作用
发布时间：2019-06-13 07:47
【摘要】：青光眼是一种以视神经损害伴进行性视野缺损为主要特征的疾病,已成为全球第二大致盲眼病。流行病学调查资料显示,我国青光眼的患病率为1.68%。在青光眼的众多分类中,原发性开角型青光眼(primary open-angle glaucoma, POAG)占据着较大比例。 迄今为止,POAG其病因和发病机制尚未完全阐明。但高眼压是青光眼性视神经损伤的主要危险因素已被广泛认可。其中眼小梁通道对房水排出的阻力增加是青光眼眼压升高的主要原因之一。小梁通道中小梁网细胞(trabecular meshwork, TM),近管结缔组织(juxtacanalicular tissue, JCT)以及Schlemm氏管内壁内皮细胞是形成房水外流阻力的主要部位,而这些组织细胞均表达丰富的肌动蛋白。肌动蛋白构形及细胞与细胞之间的连接、细胞与其周围细胞外基质的连接决定了小梁通道的构形,其结构和分布的改变可引起小梁通达对房水排出阻力的变化。 本研究选择可以作用于细胞及组织间肌动蛋白的选择性蛋白激酶抑制剂,临床已应用的药物盐酸法舒地尔(Fasudil Hydrochloride,简称Fasudil)作为处理因素。研究其作用于体外培养的牛眼小梁细胞及离体牛眼,应用形态学、免疫组织化学、分子生物学等方法分别检测药物作用前后离体牛眼房水流畅率及形态学变化；及牛眼小梁细胞形态、骨架蛋白排列及表达量的变化等。将二者结合起来以评价Fasudil对眼房水流出道是否能够减低眼房水外流阻力,增加房水流出,并且通过哪些机制调节流出道的阻力改变。从而为研究及探索原发性开角型青光眼的治疗提供新的思路及新的方向。 目的：研究Fasudil对于离体牛眼房水流畅率及前房角形态结构的作用,主要探讨其是否具有降低房水外流阻力,增加房水流出的作用。研究Fasudil作用于原代牛眼小梁细胞,主要探讨其对于细胞形态、骨架蛋白及细胞间连接蛋白等具有何种调节作用。 方法：第一部分：我们分别使用GPBS作为对照组、50uMFasudil作为实验组,15mmHg恒压灌注新鲜离体牛眼,测量基线值房水流畅率及加药后房水流畅率(C),计算增加的△C。并且使用红色荧光微球溶液灌注标记房水引流的流体模式,激光共聚焦显微镜观察测量红色荧光微球所示的房水有效滤过面积与房水丛内类似Schlemm氏管的百分比。研究Fasudil是否能够降低房水外流阻力,及探讨其促进房水外流的流体模式变化。 第二部分：组织贴块法培养原代牛眼小梁细胞并传代至3-5代,将细胞分为空白对照组,Fasudil组(予luM-1mM不同浓度的Fasudil,诱导0-2h)。分别进行MTT法药物细胞毒性研究,探讨Fasudil作于与眼小梁细胞的安全性。倒置显微镜观察细胞形态学变化、激光共聚焦显微镜观察细胞骨架蛋白F-Actin及细胞间连接蛋白Vinculin的表达及排列变化,从形态学及免疫组化学探讨Fasudil作于与眼小梁细胞骨架蛋白的作用。 结果：第一部分：使用Fasudil后,牛眼房水流畅率平均增加0.87±0.13ul/min/mmHg,(48.00+9.33%);而在对照眼其改变仅为0.34±0.03ul/min/mmHg(18.69±3.44%),这一差异在统计学上有显著意义(P0.01)。使用Fasudil的牛眼较对照组牛眼显示较为均匀的荧光微球分布和沿内壁更广泛的荧光微球标记。使用Fasudil的牛眼,平均有效滤过长度百分比(PEFL)(56.56±4.55%)是对照眼(13.36±3.58%)的4倍(P0.01)。 第二部分：成功培养牛眼小梁细胞,MTT法显示Fasudil干预牛眼小梁细胞2h后,未见明显细胞毒性。在形态学方面中,使用Fasudil后,lOuM-1mMFasudil均能使细胞形态局部皱缩、细长、细胞间隙增宽。在细胞骨架方面,Fasudil干预细胞后,F-Actin在细胞内解聚、重新排列及Vinculin在细胞间连接表达减少,且该作用在浓度和时间上存在双向依赖性。 结论：Fasudil作用于牛眼后,可以增加房水流畅率,从而降低房水外流阻力。对于牛眼小梁细胞,Fasudil具有减少细胞骨架蛋白磷酸化,使骨架蛋白重新排列,从而松弛细胞及细胞间连接的作用。且此作用安全无明显细胞毒性,提示其可能成为直接调节小梁途径骨架,降低房水外流阻力从而降低眼内压的一种新型抗青光眼类药物。.
[Abstract]:Glaucoma is a disease characterized by optic nerve damage with progressive visual field defect, and has become the second-largest blindness in the world. Epidemiological survey data show that the prevalence of glaucoma in China is 1.68%. In many classifications of glaucoma, primary open-angle glaucoma (POAG) occupies a larger proportion. To date, the etiology and pathogenesis of POAG has not yet been fully developed. But high intraocular pressure is the main risk factor of the optic nerve injury of the glaucoma. It can be found that the increase of the resistance to the drainage of the aqueous humor by the trabecular channel is the main cause of the increase of the intraocular pressure in the glaucoma. One. Trabecular Meshwork (TM), close-tube connective tissue (JCT) and Schlemm's inner wall endothelial cells are the main parts of the resistance to the outflow of aqueous humor. The connection between the cell and the cell, the connection between the cell and the extracellular matrix determines the configuration of the trabecular channel, and the change of its structure and distribution can cause the change of the resistance of the trabecula to the discharge of the aqueous humor. This study selected the selective protein kinase inhibitor which can act on the actin between the cells and the tissues, and the clinical application of the drug hydrochloride (Dialodil Hechloride) as the place The flow rate and morphological changes of the in-vitro bovine eye and aqueous humor were measured by morphological, immunohistochemical and molecular biological methods. The cell morphology, the arrangement of the protein and the amount of expression. Change and so on. Combine the two to evaluate whether the water flow out of the eye room can reduce the flow resistance of the water flow in the eye room, increase the water flow out of the room, and adjust the resistance of the outflow channel through which mechanisms The change of force is a new way to study and explore the treatment of primary open-angle glaucoma. Objective: To study the effect of Dialodil on the flow rate of aqueous humor and the morphological structure of the anterior chamber, and to study whether it has the effect of reducing the flow resistance of the aqueous humor and increasing the aqueous humor. The effect of Dialodil on the primary bovine trabecular meshwork was studied. Methods: The first part: The first part: we used the GBS as the control group and 50 uMFasaudil as the experimental group. The 15 mmHg constant pressure was used as the fresh in-vitro bovine eye. The flow rate and the post-dosing room of the baseline value were measured. The flow rate (C) of the water was calculated, and the increased flow rate C was calculated. The fluid pattern of the aqueous drainage of the aqueous humor was filled with a red fluorescent microsphere solution. The effective filtration area of the aqueous humor as shown in the red fluorescent microsphere was observed by the laser confocal microscope. The study was to study whether the flow resistance of the aqueous humor can be reduced by the study of the volume of the aqueous humor, and to explore the effect of the study on the outflow resistance of the aqueous humor. The second part: the tissue patch method was used to culture the primary bovine trabecular meshwork and subcultured to 3-5 generations, and the cells were divided into a blank control group, a Dialodil group (to luM-1 mM of various concentrations of Ruddi, 1. Induction of 0-2 h). The safety of the trabecular meshwork cells was observed. The morphological changes of the cells were observed by an inverted microscope. The expression and arrangement of the cytoskeleton protein F-Actin and the intercellular adhesion protein (Vinculin) were observed by a laser confocal microscope. Results: The first part: The flow rate of aqueous humor was increased by 0.87-0.13 ul/ min/ mmHg (48.00 + 9.33%) in the first part. The change of the control eye was only 0.34-0.03ul/ min/ mmHg (18.69-3.44%), and the difference was statistically significant. Significant significance (P0.01). A relatively uniform distribution of the fluorescent microspheres and along the inside of the eye of the control group was shown in the bulleye of Dialodil. The wall was labeled with a more extensive fluorescent microsphere. The average effective filtration length percentage (PEFL) (56.56% 4.55%) was the control eye (13.36% 3.58%) using Dialodil's bovine eye. 4-fold (P0.01). The second part: successfully cultured bovine trabecular meshwork, and the MTT method showed that the microbeam of the eye-eye was small and thin. No significant cytotoxicity was observed after the cell for 2 h. In the aspect of morphology, the cellular morphology was enabled by lOuM-1 mMFasaudil after use of Dialodil. in that cytoskeleton, the F-Actin depolymerize in the cell, the rearrangement and the decrease of the expression of Vinculin in the cell, and the effect is There was a two-way dependence on the concentration and time. The water flow rate is reduced, so that the flow resistance of the aqueous humor is reduced. So as to relax the role of cell and cell connection.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R775
本文编号：2498374
[Abstract]:Glaucoma is a disease characterized by optic nerve damage with progressive visual field defect, and has become the second-largest blindness in the world. Epidemiological survey data show that the prevalence of glaucoma in China is 1.68%. In many classifications of glaucoma, primary open-angle glaucoma (POAG) occupies a larger proportion. To date, the etiology and pathogenesis of POAG has not yet been fully developed. But high intraocular pressure is the main risk factor of the optic nerve injury of the glaucoma. It can be found that the increase of the resistance to the drainage of the aqueous humor by the trabecular channel is the main cause of the increase of the intraocular pressure in the glaucoma. One. Trabecular Meshwork (TM), close-tube connective tissue (JCT) and Schlemm's inner wall endothelial cells are the main parts of the resistance to the outflow of aqueous humor. The connection between the cell and the cell, the connection between the cell and the extracellular matrix determines the configuration of the trabecular channel, and the change of its structure and distribution can cause the change of the resistance of the trabecula to the discharge of the aqueous humor. This study selected the selective protein kinase inhibitor which can act on the actin between the cells and the tissues, and the clinical application of the drug hydrochloride (Dialodil Hechloride) as the place The flow rate and morphological changes of the in-vitro bovine eye and aqueous humor were measured by morphological, immunohistochemical and molecular biological methods. The cell morphology, the arrangement of the protein and the amount of expression. Change and so on. Combine the two to evaluate whether the water flow out of the eye room can reduce the flow resistance of the water flow in the eye room, increase the water flow out of the room, and adjust the resistance of the outflow channel through which mechanisms The change of force is a new way to study and explore the treatment of primary open-angle glaucoma. Objective: To study the effect of Dialodil on the flow rate of aqueous humor and the morphological structure of the anterior chamber, and to study whether it has the effect of reducing the flow resistance of the aqueous humor and increasing the aqueous humor. The effect of Dialodil on the primary bovine trabecular meshwork was studied. Methods: The first part: The first part: we used the GBS as the control group and 50 uMFasaudil as the experimental group. The 15 mmHg constant pressure was used as the fresh in-vitro bovine eye. The flow rate and the post-dosing room of the baseline value were measured. The flow rate (C) of the water was calculated, and the increased flow rate C was calculated. The fluid pattern of the aqueous drainage of the aqueous humor was filled with a red fluorescent microsphere solution. The effective filtration area of the aqueous humor as shown in the red fluorescent microsphere was observed by the laser confocal microscope. The study was to study whether the flow resistance of the aqueous humor can be reduced by the study of the volume of the aqueous humor, and to explore the effect of the study on the outflow resistance of the aqueous humor. The second part: the tissue patch method was used to culture the primary bovine trabecular meshwork and subcultured to 3-5 generations, and the cells were divided into a blank control group, a Dialodil group (to luM-1 mM of various concentrations of Ruddi, 1. Induction of 0-2 h). The safety of the trabecular meshwork cells was observed. The morphological changes of the cells were observed by an inverted microscope. The expression and arrangement of the cytoskeleton protein F-Actin and the intercellular adhesion protein (Vinculin) were observed by a laser confocal microscope. Results: The first part: The flow rate of aqueous humor was increased by 0.87-0.13 ul/ min/ mmHg (48.00 + 9.33%) in the first part. The change of the control eye was only 0.34-0.03ul/ min/ mmHg (18.69-3.44%), and the difference was statistically significant. Significant significance (P0.01). A relatively uniform distribution of the fluorescent microspheres and along the inside of the eye of the control group was shown in the bulleye of Dialodil. The wall was labeled with a more extensive fluorescent microsphere. The average effective filtration length percentage (PEFL) (56.56% 4.55%) was the control eye (13.36% 3.58%) using Dialodil's bovine eye. 4-fold (P0.01). The second part: successfully cultured bovine trabecular meshwork, and the MTT method showed that the microbeam of the eye-eye was small and thin. No significant cytotoxicity was observed after the cell for 2 h. In the aspect of morphology, the cellular morphology was enabled by lOuM-1 mMFasaudil after use of Dialodil. in that cytoskeleton, the F-Actin depolymerize in the cell, the rearrangement and the decrease of the expression of Vinculin in the cell, and the effect is There was a two-way dependence on the concentration and time. The water flow rate is reduced, so that the flow resistance of the aqueous humor is reduced. So as to relax the role of cell and cell connection.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R775
【参考文献】
相关期刊论文 前7条
1 徐利保;王天晓;孙薇;杨晓虹;周小平;徐岩;;Rho激酶抑制剂的研究进展[J];国际药学研究杂志;2008年06期
2 刘旭阳,王宁利,Paul Kaufman;外酶C3转移酶转基因表达对小梁细胞的影响[J];中国生物工程杂志;2005年08期
3 张青蔚;刘旭阳;吉昂;王宁利;;人眼小梁细胞的体外培养与细胞骨架和细胞连接的研究[J];眼科新进展;2006年09期
4 张强;王强;;肿瘤坏死因子-α对体外培养牛眼小梁细胞纤维连接蛋白的影响[J];眼科新进展;2009年11期
5 钟丽春,李美玉;牛眼小梁细胞培养、形态和骨架超微结构及动力学研究[J];眼科学报;1999年02期
6 郑惠珍,赵克森,黄巧冰;Rho激酶在烧伤大鼠血清诱导的血管内皮细胞骨架变化中的作用[J];中华烧伤杂志;2005年03期
7 贺翔鸽,李美玉;人眼小梁细胞培养及免疫组化特性研究[J];中华眼科杂志;1998年04期
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