牛眼小梁网细胞体外培养及吞噬功能的研究

目的研究小梁细胞吞噬功能异常在青光眼发病中的作用。方法利用牛眼分离小梁网组织，进行小梁细胞体外培养；应用乳胶微球为标记物，定性及定量观察体外培养的牛小梁细胞吞噬功能及其影响因素。结果免疫组织化学染色证实牛小梁细胞能分泌纤维连接蛋白和层连蛋白。在体外，牛小梁细胞对乳胶微球有较强的吞噬能力。细胞吞噬乳胶微球的数目随培养时间的延长而增多，培养至２４小时，平均每个细胞含４０．５±６．７个微球，细胞在吞噬过程中出现明显的形态学改变，同时也伴有自身损伤，甚至死亡。肾上腺素及可的松抑制小梁细胞的吞噬功能（Ｐ＜０．０５），ＥＧＦ对细胞吞噬作用无影响。结论小梁细胞的吞噬功能对保持房水流出道通畅起重要作用，小梁细胞的吞噬功能异常可能参与原发性开角型青光眼的发病过程。     

体外培养人眼小梁细胞吞噬乳胶微粒的研究

原发性开角型青光眼及某些继发性开角型青光眼的病理改变与覆盖在小梁柱表面的小梁细胞功能改变有关［１］；小梁细胞具有增殖、移行、吞噬及合成分泌细胞外基质、激素或酶的功能。其中小梁细胞的吞噬功能在清除异物、保持房水滤道通畅方面具有重要作用［２，３］。我们采用体外培养的人眼小梁细胞，观察其吞噬乳胶微粒的过程。一、资料和方法１小梁细胞原代和传代培养：取各种原因死亡８ｈ的新生儿眼球，按王林等［４］报道的方法进行组织块培养。无菌显微镜下操作，取出小梁组织，剪成１ｍｍ×２ｍｍ组织块，置入培养瓶内，待贴壁后加入…     

体外培养的兔虹膜色素上皮细胞吞噬功能及细胞连接的观察

目的;观察体外培养和虹膜色素上皮（iris pigment epithelium IPE）细胞是否具有吞噬功能及屏障作用。方法：将鸡红细胞悬液加入体外培养的IPE细胞内,计算其吞噬数量,电镜下观察IPE细胞间连接。结果：体外培养状态下,IPE细胞对鸡红细胞的吞噬率为5．4％,视网膜色素上皮（retinal pigment epithelium RPE)细胞为8．8％（P＜0．01）;IPE同RPE一样具有紧密连接结构。结论：兔IPE在体外培养状态下,具有吞噬功能及屏障作用。     

氧化应激对小梁网的损伤

青光跟是一组以视网膜神经节细胞(RGCs)丢失和视野缺损为特征的神经退行性疾病,其病理机制尚不完全清楚,眼压升高被认为是青光眼发生和发展最主要的危险因素.小梁网及Schlemm管是房水引流系统的主要组成部分,其结构或功能异常可引起房水流出的受阻进而引起眼压升高.越来越多的证据表明,氧化损伤可能在人小梁网细胞凋亡、功能障碍及其他退行性改变过程中发挥作用.氧化损伤是体内氧化和抗氧化失衡,从而引起脂质过氧化反应、蛋白质变性、DNA损伤等一系列组织病理损伤的过程.既往研究发现青光眼患者房水中氧化应激标志物水平升高,且氧化应激可引起小梁网细胞DNA氧化损伤、细胞内线粒体氧化损伤和炎症反应.本文就氧化应激在小梁网功能损伤中发挥的作用及可能的机制进行综述.     

小梁细胞吞噬功能的研究

前房内碎屑的沉积,如白内障囊外摘除术后晶体碎片,色素性青光眼色素、剥脱性物质、色素膜炎渗出物,前房积血的血细胞及其产物等,其清除过程均有小梁吞噬细胞参与。吞噬功能对保持房水外流系统的正常具有重要作用。现将有关小梁细胞吞噬功能研究的文献综述如下: 一、小梁细胞的吞噬功能 Rohen等证实猕猴的小梁细胞具有吞噬功能,以后人眼,其它种属猴,家兔和鼠的小梁细胞也都被发现具有吞噬活动,小梁细胞可清除房水循环中的碎片,并预防碎片对房水流出通道的阻塞。Bill认为     

小梁细胞的培养与原发性开角型青光眼的病因研究

原发性开角型青光眼（ＰＯＡＧ）是青光眼主要致盲性眼病之一，其发病机制尚不清楚。近年来许多研究已表明，ＰＯＡＧ眼压升高是由于房水排出道的病变，使房水排出阻力增加所致。阻力的部位主要在小梁网，特别是近管组织。阻力的形成与衬覆在小梁柱表面的小梁细胞形态与功能有关。由于直接研究体内小梁细胞非常困难，国外许多学者用小梁细胞体外培养的方法，研究小梁细胞的形态结构与功能活动，发现ＰＯＡＧ的发生与小梁细胞的异常密切相关，小梁细胞体外培养是探索ＰＯＡＧ病因和发病机制的有效途径。     

地塞米松对小梁细胞粘附及吞噬功能的影响

目的：探讨地塞米松对小梁细胞的粘附及吞噬功能的影响。方法：不同浓度地塞米松(10^-7M，10^-6M，10^-5M)处理体外培养的牛眼小梁细胞3d，消化、漂洗后加入用不同的细胞外基质(Extracellular matrix，ECM)包被的培养板中，37℃孵育90min后．采用甲基噻唑基四唑(Methyl thiazolyl tetrazolium，MW)法测定各组吸光度值反应粘附细胞的量；另外，以乳胶微球为标记，观察不同浓度的地塞米松对小梁细胞吞噬功能的影响。结果：与对照组比较，三种浓度的地塞米松均对小梁细胞与ECM粘附有抑制作用．且呈浓度依赖性；对小梁细胞的吞噬功能亦呈现浓度依赖性抑制作用。结论：地塞米松对小梁细胞的粘附及吞噬功能有抑制作用，抑制小梁细胞的粘附及吞噬功能可能是皮质类固醇性青光眼的发病原因之一。     

掺钕─钇铝石榴石激光虹膜切开术后房角结构改变及眼压变化

对16只青紫蓝兔32只眼行掺钕-钇铝石榴石（Nd：YAG）激光虹膜切开术，借助光镜、电镜观察术后即刻至8个月房角结构的改变。同时，临床观察术前后眼压的变化。结果显示，Nd：YAG激光虹膜切开术后，房角被纤维蛋白渗出物、组织碎屑、色素颗粒及红细胞吞噬时间推移通过房水引流即小梁滤过及细胞吞噬消化得以逐渐清除。术后一过性眼压升高可能与房角小梁网机械性堵塞有关，而非激光能量直接损伤小梁网所致。     

小梁细胞的培养与原发性开角型青光眼的病因研究

原发性开角型青光眼（ＰＯＡＧ）是青光眼主要致盲性眼病之一，其发病机制尚不清楚。近年来许多研究表明，ＰＯＡＧ眼压升高是由于房水排道的病变，使房水排出阻力增加所致，阻力的部位主要在小梁网，特别是近管组织，阻力的形成与衬覆在小梁柱表面的小梁细胞形态与功能有关。由于直接研究体内小梁细胞非常困难，国外许多学用小梁细胞体外培养的方法，研究小梁细胞的形态结构与功能活动，发现ＰＯＡＧ的发生与小梁细胞的异常密度相     

糖皮质激素性青光眼患者小梁细胞体外培养和超微结构研究

目的 探索糖皮质激素性青光眼患者小梁细胞体外培养方法及超微结构的特点。方法 从小梁切除术中取得的巩膜内板层,应用组织学培养方法进行患者小梁细胞体外培养及鉴定,并将糖皮质激素性青光眼患者小梁细胞与正常小梁细胞的超微结构进行分析和比较。结果 糖皮质激素性青光眼患者小梁组织培养的细胞经鉴定确为小梁细胞,但与正常小梁细胞相比,其细胞的微绒毛、吞饮小泡及胞浆的溶酶体含量较少。结论 糖皮质激素性青光眼患者小梁     

选择性牛眼上皮样小梁细胞的体外培养

目的选择性行牛眼上皮样小梁细胞的体外培养，为进一步研究原发性开角型青光眼提供实验材料。方法利用组织块法进行小梁细胞的原代培养，之后利用机械划除法、酶消化法、反复贴壁法，对牛眼上皮样小梁细胞进行选择性培养和传代。结果成功选择性培养出形态、性状良好的牛眼上皮样小梁细胞。结论选择性牛眼上皮样小梁细胞的体外培养所获细胞形态单一，可以用此种方法进行牛小梁细胞的体外培养并为实验应用做好准备。     

Lysosomal enzyme activities in cultured trabecular-meshwork cells

We examined the lysosomal enzyme levels in cultured trabecular meshwork cells. Histochemical studies revealed that bovine trabecular meshwork cells stained prominently for acid lipase, acid phosphatase, and acid esterase activities. Following phagocytic challenges, such as incubations with latex microspheres or zymosan particles, lysosomal enzyme staining was visibly enhanced. The uptake of foreign particles by trabecular-meshwork cells was evident. Data from biochemical assays for acid lipase, cholesteryl esterase, and acid esterase confirmed the histochemical findings. These results indicate that trabecular meshwork cells are actively phagocytic in culture and that lysosomal enzyme levels in these cells may be modulated by phagocytic challenge.     

The trabecular meshwork in acute and chronic angle closure glaucoma

PURPOSE: To determine the effect of acute and chronic primary angle closure glaucoma (PACG) on the trabecular meshwork. METHODS: Trabecular specimens of 16 consecutive patients with primary angle closure glaucoma (PACG)--6 acute PACG eyes, and 10 chronic PACG eyes without an acute attack--were studied by light and electron microscopy. RESULTS: Acute PACG: The trabecular meshwork revealed a generalised oedema and an accumulation of pigment in the widened trabecular spaces and Schlemm's canal. Attenuated trabecular endothelial cells appeared to be devoid of subcellular components. Chronic PACG: In chronic PACG eyes the trabecular architecture had lost its regular arrangement, with fewer and narrower trabecular spaces and fusion of the trabecular beams in areas. There were numerous electron-dense bodies in the trabecular tissues, both within the trabecular beams and in the extracellular spaces, which had a banded fibrillar structure. An overall loss of endothelial cells was noted; the remaining cells were crowded together and were polymorphic. Melanin pigment was present both within the stroma and in the endothelial cells. CONCLUSIONS: Pigment accumulation in the trabecular spaces and within the cells and a noninflammatory degeneration appeared to be the primary changes in the trabecular meshwork after acute angle closure glaucoma. In chronic PACG eyes, there was evidence of loss of endothelial cells and reactive repair processes. These changes were present in areas away from visible peripheral anterior synechiae. A gonioscopic evaluation of the extent of peripheral anterior synechiae alone may not reflect the extent of trabecular meshwork damage in acute and chronic PACG. Patients experiencing an acute attack of PACG require a long-term follow up, because the intraocular pressure (IOP) may rise later, due to ongoing changes compromising the outflow facility, or due to the effects of aging in the trabecular meshwork.     

Decompensation of chronic open-angle glaucoma following mydriasis-induced pigmentary dispersion into the aqueous humour: a light and electron microscopic study.

A patient with well controlled chronic open-angle glaucoma developed after dilatation of both pupils persistent increase in intraocular pressure (IOP) due to extensive pigmentary dispersion into the aqueous humour. Trabeculectomy specimens obtained from both eyes after 3 and 7 weeks were studied by light and electron microscopy. It seems evident that the initial phase of raised IOP was caused by a clogging mechanism to the outflow channels by melanin and phagocytic cells. The permanent increase in IOP is attributed to the damage induced in the fibrous components of the trabecular sheets as a result of a complete breakdown of their endothelial covering.     

地塞米松诱导牛眼小梁细胞凋亡的研究

目的：探讨地塞米松诱导牛眼小梁细胞凋亡的机制。方法：采取体外组织块培养法,培养新生牛眼小梁细胞。将1－500mg/L地塞米松加入融合的第3－5代小梁细胞培养液中,作用1－14d,用相差显微镜、荧光显微镜、透射电子显微镜、DNA电泳及流式细胞仪检测细胞凋亡情况。结果：125－500mg/L地塞米松作用1－14d,小梁细胞凋亡呈浓度时间依赖性。 凋亡的小梁细胞经Hoechst3258染色,于荧光显微镜下观察,细胞核呈致密度浓染的颗粒块状荧光;透射电镜下可见小梁细胞染色质固缩,并有凋亡小体,细胞器基本完好;DNA电泳呈梯状条带,流式细胞仪测到亚二倍体峰。结论：地塞米松可诱导体外培养的小梁细胞凋亡,可能是激素性青光眼的发病机制之一。     

Histologic findings in pigment dispersion syndrome and pigmentary glaucoma

PURPOSE: To investigate the morphologic changes in the trabecular meshwork in a case series of eyes with pigment dispersion syndrome and pigmentary glaucoma, and surgical trabeculectomy specimens from eyes with pigmentary glaucoma. MATERIALS AND METHODS: Trabecular meshworks from 6 whole eyes from 3 donors and 7 trabeculectomy specimens were studied by light and electron microscopy. Axonal counts from the whole eyes were correlated with qualitative and quantitative data of meshwork changes. RESULTS: Changes in the meshwork varied around the circumference of the eyes, but in all 6 eyes in most regions of the circumference there were numerous pigment granules within trabecular cells; pigment was not found within intertrabecular or cribriform spaces. In some regions of the circumference there was trabecular cell loss, loss of intertrabecular spaces, fusion of lamellae, and an increase in extracellular material under the inner wall of the canal. Separation of the normal tendinous connection to the canal wall cells was noted in some regions of all eyes. This change could be associated with regions of pathologic separation of the inner wall from the cribriform region, associated with partial obliteration of the lumen of the canal with cells and cell processes. In eyes with pronounced axon loss, meshworks showed most pronounced loss of trabecular cells and increased extracellular material. Trabeculectomy specimens had similar changes and, in addition, showed damaged trabecular cells and collapse of intertrabecular spaces without fusion of lamellae, consistent with artifacts from manipulation during surgery. CONCLUSIONS: Loss of trabecular cells, fusion of trabecular lamellae with collapse of intertrabecular spaces, increase in extracellular material, and obliteration of the canal were found in various amounts around the circumference of eyes with pigment dispersion syndrome and elevated intraocular pressure, and pigmentary glaucoma. These probably all contribute to the development of increased intraocular pressure. Meshworks from trabeculectomy specimens showed these findings and also showed artifactual damage of cells and loss of intertrabecular spaces. This suggests that handling during surgery may cause single trabeculectomy specimens to give only an incomplete picture of the pathophysiology of pigmentary glaucoma.     

Ghost cells as a cause of glaucoma.

Clinical and investigative evidence indicated a glaucoma caused mainly by degenerated red blood cells, or ghost cells. These ghost cells, with altered shape, color, and pliability, accumulated in the vitreous cavity after hemorrhage. Following disruption of the anterior hyaloid face, they passed into the anterior chamber and caused severe glaucoma. In the anterior chamber, the tiny, khakicolored cells, circulating slowly, were frequently mistaken for white blood cells. They covered the trabecular meshwork or filled the inferior angle with a pathognomonic khaki-colored layer. They were identified by phase-contrast microscopic examination of anterior chamber aspirates. The decreased pliability of these degenerated cells seemed to account for their inability to pass easily through the human trabecular meshwork and, therefore, to cause severe glaucoma.     

Glaucomatous outflow pathway and oxidative stress

Oxidative free radicals and reactive oxygen species (ROS) are able to affect the cellularity of the human trabecular meshwork (HTM). These findings suggest that intraocular pressure increase, which characterises most glaucomas, is related to oxidative degenerative processes affecting the HTM and specifically its endothelial cells. Much evidence indicates that in this region ROS play a fundamental pathogenic role by reducing local antioxidant activities, inducing outflow resistance and exacerbating the activities of superoxide dismutase and glutathione peroxidase in glaucomatous eyes. Furthermore, hydrogen peroxide induces rearrangement of HTM cells and compromises their integrity. Glaucomatous subjects might have a genetic predisposition rendering them more susceptible to ROS-induced damage. A fairly significant correlation between oxidative DNA damage in the HTM and intraocular pressure increase and visual field defects in glaucomatous patients has been demonstrated. Thus, oxidative stress may play a significant role during glaucoma course initially damaging HTM cells, then contributing to the alteration of the homeostasis between NO and endothelins, and finally through its possible involvement in ganglional cell death. On the whole, these findings support the hypothesis that oxidative damage is an important step in the pathogenesis of primary open-angle glaucoma, and might be a relevant target for both prevention and therapy.     

小梁网的干细胞移植治疗原发性开角型青光眼的研究进展

原发性开角型青光眼(primary open angle glaucoma,POAG)是以持续性眼压增高导致视神经损伤为主要临床表现的一种疾病,其发病机制复杂,尚未明确,现阶段临床治疗相对困难。影响眼内压(intraocular pressure,IOP)高低的重要因素是房水引流是否通畅,而房水引流途径中小梁网(trabecular meshwork,TM)起重要调控作用。TM细胞的形态、数量、结构和功能改变均可使房水外流阻力增大,从而导致IOP升高。研究证实诱导多功能干细胞(induced pluripotent stem cells,iPSCs)、骨髓间充质干细胞(bone mesenchymal stem cells,BMSCs)和脂肪干细胞(adipose-derived stem cells,ADSCs)已被用于TM细胞的分化和再生,为POAG小梁网的干细胞替代治疗提供可靠的细胞来源。近年研究发现,小梁网干细胞(trabecular meshwork stem cells,TMSCs)在分化为TM细胞方面具有绝对优势,为细胞移植治疗青光眼提供新的靶向,这标志着干细胞治疗POAG进入一个新纪元,为青光眼治疗带来新的曙光。本文将对不同种类干细胞的小梁网移植进行综述,为细胞移植治疗POAG提供新思路。