葡萄糖对POAG患者小梁网细胞凋亡的影响

目的探讨不同浓度葡萄糖对POAG患者小梁网细胞凋亡的影响,研究葡萄糖与POAG以及糖尿病与POAG之间的关系,进一步探讨POAG的发病机制。方法实验研究。采用组织块培养法原代培养POAG患者小梁网细胞,取传代的小梁网细胞分别加入含葡萄糖终浓度为0.0（对照组）、5.5、45.0 mmol/L的无血清培养基,分别培养48 h后,采用CCK-8和流式细胞仪法检测不同浓度葡萄糖对小梁网细胞的影响。数据采用单因素方差进行分析。结果通过CCK-8检测发现,随着浓度的增加,葡萄糖对小梁网细胞的增殖抑制作用增强,可促进细胞凋亡,且各实验组与对照组之间相比差异有统计学意义（F=13.87,P＜0.01）;通过流式细胞仪法检测葡萄糖浓度为5.5、45.0 mmol/L实验组小梁网细胞凋亡率分别为10.65%±0.03%、25.74%±0.02%,均高于对照组（4.02%±0.03%）,且差异有统计学意义（F=25.34,P＜0.01）。结论高糖可以使小梁网细胞的增殖能力下降,凋亡率增加,从而可能导致小梁网细胞网状结构改变,房水流出途径的阻力增加。     

整合素在人眼小梁网组织中的表达及意义的研究进展

整合素是一类细胞膜表面的糖蛋白受体家族分子，在小梁网组织中表达多种整合素亚单位。可以和细胞外基质结合并调节基质金属蛋白酶的表达。影响小梁网内环境的稳定，改变房水外流阻力。本文就整合素与小梁网组织的关系作一综述。     

体外培养猴眼小梁细胞及睫状肌细胞中组织金属蛋白酶抑制剂的表达

目的：观察体外培养的正常猴眼小梁细胞及睫状肌细胞中组织金属蛋白酶抑制剂（tissue inhibitors of metalloproteinase,TIMP)的表达,探讨生理状态下基质金属蛋白酶（matrix metalloproteinase,MMP)及TIMP在小梁网房水流出及葡萄膜－巩膜房水流出的通道中的作用。方法：采用Reverse-zymography技术,检测猴眼小梁细胞及睫状肌细胞中TIMP及MMP的表达。结果;猴眼小梁细胞及睫状肌细胞培养液中均可见TMIP－1、TIMP－2及TIMP－3表达,小梁细胞中MMP／TIMP比值明显高于睫状肌细胞。结论：正常状态下小梁细胞外基质的降解能力明显高于睫状肌细胞,可能是由于传统的小梁网房水流出通道作用强于葡萄膜－巩膜房水流出通道 的作用。     

广谱机械力敏感离子通道抑制剂钌红调控眼压的实验研究

目的 研究广谱机械力敏感离子通道抑制剂钌红对眼压的调控作用。设计 实验研究。研究对象 人原代小梁网细胞、C57BL/6J小鼠（2月龄）10只。方法 分离培养人原代小梁网细胞，通过细胞形态和免疫荧光技术鉴定细胞类型；利用膜片钳技术记录机械力刺激下小梁网细胞机械力敏感电流的变化，并观察广谱机械力敏感离子通道抑制剂钌红对电流的抑制作用；将10只小鼠随机分为2组（每组5只）：第1组（对照组）小鼠双眼前房内分别注射2 μl生理盐水，第2组（钌红组）小鼠双眼前房内分别注射2 μl 20 μM钌红。注射前日测量基础眼压，注射后每日测量眼压，并于注射12天后在小鼠每眼的前房设定15、25、35 mmHg压力下各灌注15 min生理盐水的方式测量房水流畅系数及注入速率。主要指标 机敏电流（pA）、眼压（mmHg）和房水流畅系数（μl/min/mmHg）。结果 钌红组给药后机械力敏感电流（-67.6 ± 30.2 pA）显著低于给药前（-309.7 ± 65.9 pA）（P=0.003）；注射10天后，钌红组眼压为（14.67±2.11）mmHg，低于生理盐水组（17.85±3.66 mmHg）（P=0.0604）；注射12天后，钌红组房水流畅系数（0.01058±0.00236 μl/min/mmHg）明显低于生理盐水组（0.00324 ± 0.00135 μl/min/mmHg）（P=0.0273）；灌注压为15 mmHg时，钌红组注入速率（0.37±0.15 μl/min）高于生理盐水组（0.28±0.05 μl/min）（P=0.6278）。结论 钌红有效抑制人小梁网细胞机械力敏感电流，降低小梁网房水引流功能；同时，钌红也可有效抑制房水生成速率，为青光眼治疗提供新思路。     

细胞骨架作用剂抗青光眼的研究进展

细胞骨架与细胞的各种活动和功能密切相关。在细胞外基质的参与下，细胞骨架调控着房水流出通道的解剖学构架，从而影响小梁网的滤过功能。研究表明，细胞骨架作用剂，如利尿酸、latrunculins、细胞松弛素、某些蛋白激酶抑制剂（H-7、HA1077、Y-27632）、blebbistatin、他汀类药物和BDM均可增加房水流出易度。目前的研究正在致力于探讨细胞骨架作用剂调节房水流出的机制。种种迹象表明，细胞骨架作用剂有可能成为有潜力的抗青光眼药物。     

青光眼的基因治疗：现状与挑战

原发性青光眼是受环境因素影响的多基因复杂遗传病，针对青光眼的基因治疗策略尚不能仅单纯使用针对单一致病基因进行矫正或补偿，但目前基因疗法仍可显示出积极的延缓发病、减轻或改善症状等“治标”结果。目前的青光眼基因治疗策略包括以增加房水排出为主的靶向小梁网的细胞骨架调节蛋白及靶向葡萄膜巩膜房水排出通道的基因疗法，以及以减少房水生成为主的针对睫状体上皮的基因疗法等。此外还有滤过性手术后抗瘢痕的基因疗法（如术后将基质金属蛋白酶3基因转入术区表达）、视网膜神经保护基因疗法[在内层视网膜表达脑源性神经营养因子、激活神经营养素受体等而改善视网膜神经节细胞（RGC）营养，外源表达抗凋亡基因产物以拮抗RGC凋亡，免疫调节减轻RGC凋亡，调节胶质细胞功能支持RGC，减少氧化应激活性氧抗RGC凋亡等]。但目前的病毒载体仍有一定几率的风险性，通过改造转基因载体使其实现精准、特异的靶向表达是一种有效解决办法，这也是推动青光眼基因疗法技术实用化的关键。此外，如何能维持长效表达增加有效治疗期也是青光眼基因治疗面临的挑战。（眼科，2022，31： 1-7）     

白细胞介素-1α对猪小梁细胞基质金属蛋白酶／基质金属蛋白酶抑制剂表达的影响

背景房水外流通路的阻塞或小梁网细胞外基质（ECM）的异常堆积导致房水流畅系数降低是引起眼压升高的原因之一,基质金属蛋白酶（MMPs）和组织金属蛋白酶抑制剂（TIMPs）的平衡对ECM的代谢至关重要,白细胞介素-1α（IL-1α）可以通过调节MMPs的表达而调节房水的外流。目的研究猪重组IL-1α对体外培养的猪眼小梁细胞MMP-2、MMP-3及TIMP-1表达的影响。方法从猪眼取出带有小梁组织的巩膜,用组织块培养法培养小梁细胞并进行传代,第3代的猪小梁细胞应用纤维连接蛋白（FN）和层黏连蛋白（LM）进行鉴定。第3代小梁细胞血清饥饿培养24h后分为2组,对照组加入无血清培养基,IL组加入10m／L IL-1α,分别培养30min。采用细胞免疫化学法分析IL组MMP-2、MMP-3及TIMP-1蛋白在培养小梁细胞中的表达;采用逆转录聚合酶链反应（RT—PCR）法检测小梁细胞中MMP-2mRNA、MMP-3mRNA及TIMP-1 mRNA的表达,并与对照组的检测结果进行比较。结果传3代的细胞对FN和LM呈现阳性反应。与对照组比较,IL组小梁细胞中MMP-3和TIMP-1蛋白的表达量（A值）明显升高,差异均有统计学意义（t=-7．694、t=-5．199,P〈0．05）,但2组间小梁细胞中MMP-2表达的差异无统计学意义（t=-2．365,P〉0．05）。IL组小梁细胞中MMP-3mRNA和TIMP-1mRNA的表达量（A值）明显高于对照组,差异均有统计学意义（t=-3．025、t=-1．921,P〈0．05）,而2组间小梁细胞中MMP-2mRNA的表达差异无统计学意义（t=-1．173,P〉0．05）。结论外源性IL-1α能增加猪眼小梁细胞中MMP-3、TIMP-1的表达,引起MMP-3／TIMP-1平衡改变,促进小梁网ECM的分解,增加房水外流,而对MMP-2的表达无影响。     

纤维连接蛋白与人眼小梁网组织关系的研究进展

纤维连接蛋白（fibronectin,FN）是一种具有重要生物学活性的非胶原糖蛋白,是细胞外基质（extracellular matrix．ECM）的重要组成部分,参与机体的细胞连接黏附、增殖分化、信号传导、基因表达、收缩调节、上皮组织修复、排列规则化以及机体免疫调控等重要的生理功能。在人眼小梁网组织（human trabecular meshwork,HTC）中表达丰富的FN,可和整合素（integrin）、共结合蛋白聚糖（syndecan）、转化生长因子β（transforming growth faetor-β,TGF—β）等ECM相互作用来影响细胞间扣细胞与基质间骨架的改变．最终影响小梁网内环境的稳定,改变房水外流阻力。研究表明,小梁网ECM含量的改变可使HTC网眼狭窄或塌陷,导致房水外流阻力增加,从而使眼内压升高,最终导致视功能丧失。近些年来人们对FN的结构功能、活性调节在原发性开角型青光眼的发病机制中所起的作用有了初步认识．现就FN与HTC关系的研究现状作一综述。     

非编码RNA调控小梁网组织在青光眼中作用的研究进展

小梁网是前房水引流的重要通道,呈复杂的三维结构,主要的细胞成分是小梁网细胞,细胞之间有交错成多层的细胞外基质。小梁网结构和功能的改变是导致眼压失常甚至视神经损伤的重要原因。有研究发现,非编码RNA(ncRNA)的异常表达可导致小梁网细胞生存率、收缩性能和细胞外基质结构发生改变,房水流出受阻,眼压失控,是青光眼发生的重要机制。目前,与小梁网相关的ncRNA研究涉及多种ncRNA分子和多种类型青光眼,本文将对微小RNA、长链ncRNA和环状RNA等ncRNA在小梁网组织中的研究进展进行综述。     

转化生长因子β1对体外培养的牛眼小梁细胞MMP3和MMP9表达的影响

目的 探讨转化生长因子β1（TGF-β1）对培养的牛眼小梁细胞基质金属蛋白酶（MMPs)的影响。房水引流阻力在原发性开角型青光眼（POAG）发病机制中的作用。方法 对牛眼小梁细胞进行体外原代及传代培养，对传三代小梁细胞分别施加含TGF-β1终浓度为0、0.5,1,5ng/ml的培养液，48h后行MMP3及MM农艺师免疫组人SP法染色。结果进行计算机图像分析并行统计学检验。结果 体外培养的牛眼小梁细胞表达MMP3及MMP9，TGF-β1可抑制小梁细胞MMP3及MMP9表达。结论 TGF-β1在一定范围内抑制小梁细胞MMP3及MMP9表达，造成小梁网ECM的异常堆积，导致房水引流阻力增高。     

miR-17-5p对小梁网细胞外基质蛋白表达的调控作用及其机制

目的　探讨miR-17-5p对小梁网细胞外基质（ECM）蛋白表达的调控作用及其机制。方法　收集原发性开角型青光眼（POAG）患者和正常小梁网组织,利用RT-PCR和Western blot分别检测miR-17-5p和Smad3表达;将人小梁网细胞（HTMCs）分为miR-17-5p mimics和miR-17-5p NC组,分别转染载有miR-17-5p mimics组和miR-17-5p NC的载体,Western blot检测HTMCs中ECM蛋白纤连蛋白（FN）、胶原蛋白I（CoL-I）、α-平滑肌肌动蛋白（α-SMA）的表达;靶基因预测库预测miR-17-5p靶基因,荧光素酶报告载体联合Western blot鉴定靶基因。HTMCs中共转染miR-17-5p mimics和Smad3过表达载体,Western blot检测各转染组细胞中Smad3、FN、CoL-I、α-SMA的蛋白表达。结果　正常小梁网组织中miR-17-5p表达量为1.16±0.11,高于POAG小梁网组织的0.19±0.04,差异有统计学意义（t=10.641,P<0.001）;正常小梁网组织中Smad3蛋白表达量为0.31±0.06,低于POAG小梁网组织的0.86±0.07,差异有统计学意义（t=8.105,P<0.001）。miR-17-5p mimics组HTMCs中FN、CoL-I和α-SMA蛋白表达均低于miR-17-5p NC组,差异均有统计学意义（均为P<0.001）;靶基因预测库预测及荧光素酶报告载体联合Western blot鉴定证实Smad3为miR-17-5p下游靶基因;miR-17-5p+Smad3转染组HTMCs中Smad3、FN、CoL-I和α-SMA蛋白表达均高于miR-17-5p+vector转染组,差异均有统计学意义(均为P<0.001)。结论　miR-17-5p 能抑制HTMCs的ECM蛋白表达,这一过程可能是通过靶向抑制Smad3表达而实现的。     

Effects of ML-7 and Y-27632 on carbachol- and endothelin-1-induced contraction of bovine trabecular meshwork

The trabecular meshwork is considered a smooth muscle like tissue contributing to aqueous outflow regulation and thus to regulation of intraocular pressure. An elevation in intraocular pressure is one of the greatest risk factors for most forms of glaucoma. We assume that contraction of trabecular meshwork reduces aqueous humor outflow and thus enhances intraocular pressure, whereas relaxation exerts the opposite effect. The present paper supports the hypothesis of the trabecular meshwork being a smooth muscle-like tissue. We perform measurements of isometric force in isolated bovine trabecular meshwork strips. Contractility of this tissue is induced by carbachol or endothelin-1. The contractile force is successfully inhibited by ML-7, a highly specific inhibitor of myosin light chain kinase. The contraction is also reduced in the presence of the RhoA kinase inhibitor Y-27632. We further describe the protein expression of smooth muscle myosin and its regulatory kinase, the myosin light chain kinase, in human and bovine trabecular meshwork cells. Additionally, the serine phosphorylation of myosin light chain kinase is shown. These data indicate that the trabecular meshwork expresses major contractility regulating proteins which are involved in tissue function. Inhibition of the signaling pathways which lead to myosin phosphorylation causes inhibition of contractile force in trabecular meshwork. According to our concept of aqueous humor outflow regulation, trabecular meshwork relaxing substances appear to be ideal antiglaucomatous drugs, leading to increased outflow facility.     

Effects of chemical inhibition of N-WASP, a critical regulator of actin polymerization on aqueous humor outflow through the conventional pathway

The integrity of actin cytoskeletal organization in aqueous humor outflow pathway is thought to play a critical role in modulation of aqueous humor outflow through the trabecular meshwork. Our understanding of the regulation of actin cytoskeletal dynamics in outflow pathway, however, is very limited. To explore the potential importance of Neural Wiskott-Aldrich syndrome protein (N-WASP), a critical regulator of actin polymerization/nucleation in aqueous humor outflow pathway, the effects of Wiskostatin, a selective pharmacological inhibitor of N-WASP, on aqueous humor outflow facility were evaluated using enucleated porcine eyes and a constant pressure perfusion system. Further, drug induced effects on actin cytoskeletal organization, cell adhesions, myosin II phosphorylation, matrix metalloproteinase (MMP) activity, and cytoskeletal protein profile in porcine trabecular meshwork (TM) cells were determined by immunofluorescence, zymography, and mass spectrometry. Aqueous humor outflow facility was increased significantly and progressively in the Wiskostatin perfused porcine eyes. The Wiskostatin perfused eyes appear to exhibit increased giant vacuoles in the inner wall of aqueous plexi and deformation of aqueous plexi. The Wiskostatin treated TM cells demonstrated extensive vacuoles in their cytosol, and both actin stress fibers and focal adhesions were decreased in a reversible manner. The drug-treated TM cells also revealed decreased myosin II and actin in the cytoskeletal enriched triton insoluble fraction but did not affect myosin II phosphorylation or MMP-2 activity. These data demonstrate that the chemical inhibition of N-WASP increases aqueous humor outflow facility in association with decreased actomyosin interaction and cell adhesive interactions revealing the importance of N-WASP in homeostasis of aqueous humor outflow.     

水通道蛋白l与房水的产生及排出的关系

目的 研究水通道蛋白(AQPI)在房水的产生、排出及调节机制中的作用。方法 运用免疫组织化学法测定人眼房水的产生及排出通路相关组织中AQPl的表达。结果 在睫状体非色素上皮、小梁网内皮细胞、Schlemm‘‘‘‘s管内皮细胞及虹膜基质中有AQPl的表达。结论 证实了AQPI在房水的产生及排出通路组织细胞中的表达，为房水的正常代谢及青光眼发病机制的研究提供了分子生物学基础。     

Perfusion of his-tagged eukaryotic myocilin increases outflow resistance in human anterior segments in the presence of aqueous humor

PURPOSE: A previous study by the authors has shown that recombinant myocilin purified from a prokaryotic expression system increases outflow resistance in cultured human anterior segments. The present study was performed to determine whether full-length myocilin purified from a human trabecular meshwork cell expression system alters outflow resistance after infusion into human anterior segments. METHODS: A feline immunodeficiency virus vector encoding both full-length myocilin (amino acids 1-503 fused to C-terminal V5 and six-histidine epitopes) and puromycin resistance was used to transduce a transformed trabecular meshwork cell line (TM5). Stably expressing cells were selected with puromycin. Recombinant myocilin was purified from the media using nickel ion affinity chromatography. Control purifications were performed on media from parental TM5 cells. Anterior segments of human eyes were placed in organ culture and perfused with either Dulbecco's modified Eagle's medium (DMEM) or DMEM supplemented with 50% porcine aqueous humor. One eye received an anterior chamber exchange with recombinant myocilin (2 microg/mL), whereas the fellow eye received an equal volume of control. Immunohistochemistry was performed with anti-myocilin and anti-V5 antibodies. Native polyacrylamide gel electrophoresis was used to analyze myocilin complex formation in porcine aqueous humor. RESULTS: Recombinant myocilin in porcine aqueous humor increased outflow resistance in cultured human anterior segments (91% +/- 68% [mean +/- SD] versus 18% +/- 31% in fellow control eye; n = 9, P = 0.004). Maximum outflow resistance was obtained 5 to 17 hours after infusion and remained above baseline for >3 days. Recombinant myocilin also increased outflow resistance in eyes incubated in DMEM, but only if myocilin was preincubated with porcine aqueous humor (78% +/- 77% when preincubated in DMEM containing porcine aqueous humor versus 13% +/- 15% when preincubated with DMEM alone, n = 6, P = 0.03). Recombinant myocilin appears to form a complex in porcine aqueous humor with a heat-labile protein(s). Immunohistochemistry revealed the presence of myocilin in the juxtacanalicular region of the trabecular meshwork. CONCLUSIONS: Myocilin purified from human trabecular meshwork cells increased outflow resistance in cultured human anterior segments, but only after incubation with porcine aqueous humor. Recombinant myocilin appears to form a complex in porcine aqueous humor that enables it to bind specifically within the trabecular meshwork.     

Direct involvement of trabecular meshwork in the regulation of aqueous humor outflow

Our new hypothesis for the regulation of aqueous humor outflow suggests that the trabecular meshwork is not a passive filter but an active contractile element contributing to the ciliary muscle traction affecting it. The trabecular meshwork contains contractile smooth-muscle-specific alpha-actin filaments, and its cells exhibit electrical properties typical for smooth muscle cells. Contractility measurements performed for the first time in isolated trabecular meshwork enable a functional comparison with ciliary muscle. Pharmacologic outflow regulation has been determined in isolated perfused anterior segments with intact trabecular meshwork and total absence of ciliary muscle. Substances that contracted isolated trabecular meshwork (e.g., pilocarpine) decreased the outflow rate, whereas relaxants (e.g., low-dose epinephrine) increased it. The concept of a functional antagonism between the trabecular meshwork and the ciliary muscle has to be considered.     

原发性开角型青光眼房水外流通路改变的研究进展

原发性开角型青光眼(POAG)是一种以视神经轴索及相关视网膜神经节细胞丢失为特征的视神经病变.眼压升高是POAG最重要的危险因素.大多数POAG患者眼压升高主要是房水外流阻力异常增高所致.小梁网是产生房水排出阻力的主要部位.目前多数研究者认为POAG患者小梁网功能不良与致炎因子表达、细胞老化、氧化应激损伤及细胞质成分减少等因素有关.小梁网细胞本身及细胞外基质的变化均可以引起房水外流阻力的改变,进而导致眼内压的升高.为了进一步开展对POAG发病机制的研究,有必要就目前有关POAG患者房水外流通路改变的研究进展予以综述,旨在为POAG的深入研究提供参考依据.