鹽酸法舒地爾對離體牛眼房水流暢系數(shù)及牛眼小梁細胞骨架的作用
發(fā)布時間：2019-06-13 07:47
【摘要】：青光眼是一種以視神經(jīng)損害伴進行性視野缺損為主要特征的疾病,已成為全球第二大致盲眼病。流行病學調(diào)查資料顯示,我國青光眼的患病率為1.68%。在青光眼的眾多分類中,原發(fā)性開角型青光眼(primary open-angle glaucoma, POAG)占據(jù)著較大比例。 迄今為止,POAG其病因和發(fā)病機制尚未完全闡明。但高眼壓是青光眼性視神經(jīng)損傷的主要危險因素已被廣泛認可。其中眼小梁通道對房水排出的阻力增加是青光眼眼壓升高的主要原因之一。小梁通道中小梁網(wǎng)細胞(trabecular meshwork, TM),近管結締組織(juxtacanalicular tissue, JCT)以及Schlemm氏管內(nèi)壁內(nèi)皮細胞是形成房水外流阻力的主要部位,而這些組織細胞均表達豐富的肌動蛋白。肌動蛋白構形及細胞與細胞之間的連接、細胞與其周圍細胞外基質(zhì)的連接決定了小梁通道的構形,其結構和分布的改變可引起小梁通達對房水排出阻力的變化。 本研究選擇可以作用于細胞及組織間肌動蛋白的選擇性蛋白激酶抑制劑,臨床已應用的藥物鹽酸法舒地爾(Fasudil Hydrochloride,簡稱Fasudil)作為處理因素。研究其作用于體外培養(yǎng)的牛眼小梁細胞及離體牛眼,應用形態(tài)學、免疫組織化學、分子生物學等方法分別檢測藥物作用前后離體牛眼房水流暢率及形態(tài)學變化；及牛眼小梁細胞形態(tài)、骨架蛋白排列及表達量的變化等。將二者結合起來以評價Fasudil對眼房水流出道是否能夠減低眼房水外流阻力,增加房水流出,并且通過哪些機制調(diào)節(jié)流出道的阻力改變。從而為研究及探索原發(fā)性開角型青光眼的治療提供新的思路及新的方向。 目的：研究Fasudil對于離體牛眼房水流暢率及前房角形態(tài)結構的作用,主要探討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細胞,主要探討其對于細胞形態(tài)、骨架蛋白及細胞間連接蛋白等具有何種調(diào)節(jié)作用。 方法：第一部分：我們分別使用GPBS作為對照組、50uMFasudil作為實驗組,15mmHg恒壓灌注新鮮離體牛眼,測量基線值房水流暢率及加藥后房水流暢率(C),計算增加的△C。并且使用紅色熒光微球溶液灌注標記房水引流的流體模式,激光共聚焦顯微鏡觀察測量紅色熒光微球所示的房水有效濾過面積與房水叢內(nèi)類似Schlemm氏管的百分比。研究Fasudil是否能夠降低房水外流阻力,及探討其促進房水外流的流體模式變化。 第二部分：組織貼塊法培養(yǎng)原代牛眼小梁細胞并傳代至3-5代,將細胞分為空白對照組,Fasudil組(予luM-1mM不同濃度的Fasudil,誘導0-2h)。分別進行MTT法藥物細胞毒性研究,探討Fasudil作于與眼小梁細胞的安全性。倒置顯微鏡觀察細胞形態(tài)學變化、激光共聚焦顯微鏡觀察細胞骨架蛋白F-Actin及細胞間連接蛋白Vinculin的表達及排列變化,從形態(tài)學及免疫組化學探討Fasudil作于與眼小梁細胞骨架蛋白的作用。 結果：第一部分：使用Fasudil后,牛眼房水流暢率平均增加0.87±0.13ul/min/mmHg,(48.00+9.33%);而在對照眼其改變僅為0.34±0.03ul/min/mmHg(18.69±3.44%),這一差異在統(tǒng)計學上有顯著意義(P0.01)。使用Fasudil的牛眼較對照組牛眼顯示較為均勻的熒光微球分布和沿內(nèi)壁更廣泛的熒光微球標記。使用Fasudil的牛眼,平均有效濾過長度百分比(PEFL)(56.56±4.55%)是對照眼(13.36±3.58%)的4倍(P0.01)。 第二部分：成功培養(yǎng)牛眼小梁細胞,MTT法顯示Fasudil干預牛眼小梁細胞2h后,未見明顯細胞毒性。在形態(tài)學方面中,使用Fasudil后,lOuM-1mMFasudil均能使細胞形態(tài)局部皺縮、細長、細胞間隙增寬。在細胞骨架方面,Fasudil干預細胞后,F-Actin在細胞內(nèi)解聚、重新排列及Vinculin在細胞間連接表達減少,且該作用在濃度和時間上存在雙向依賴性。 結論：Fasudil作用于牛眼后,可以增加房水流暢率,從而降低房水外流阻力。對于牛眼小梁細胞,Fasudil具有減少細胞骨架蛋白磷酸化,使骨架蛋白重新排列,從而松弛細胞及細胞間連接的作用。且此作用安全無明顯細胞毒性,提示其可能成為直接調(diào)節(jié)小梁途徑骨架,降低房水外流阻力從而降低眼內(nèi)壓的一種新型抗青光眼類藥物。.
[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.
【學位授予單位】：復旦大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R775
本文編號：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.
【學位授予單位】：復旦大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R775
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