青光眼病人小梁细胞体外培养、冻存与复苏

目的：探索青光眼病人小梁细胞体外培养和保存方法。方法：从小梁切除术取得的巩膜内板层，应用组织培养方法进行病人小梁细胞体外培养，完成鉴定工作。并将第四代的青光眼病人小梁细胞冻存，冻存２周，１个月，２个月，半年后，将细胞复苏，观察复苏后小梁细胞的生长情况。结果：青光眼病人小梁组织培养出的细胞经鉴定为小梁细胞，培养的小梁细胞经冷冻保存，复苏率超过８０％。结论：青光眼病人小梁细胞体外培养及冻存复苏成功，使     

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     

人眼小梁细胞体外培养,冻存与复苏

目的:建立人小梁细胞体外培养及对其进行冻存、复苏研究。方法:应用组织块培养方法进行人小梁细胞体外培养,将第3代的小梁细胞冻存,冻存1周、2周、1月、2月后细胞给予复苏,观察复苏后细胞的生长情况。结果:人眼小梁细胞体外培养成功。冻存的小梁细胞复苏成功,所有复苏率超过90％。结论:人眼小梁细胞体外培养及冻存复苏成功,为以后构建小梁细胞的cDNA文库,为筛选青光眼发病的相关基因提供有利的实验基础。眼科学报1998;14:73—75。     

Aqueous humor stimulates the migration of human trabecular meshwork cells in vitro

PURPOSE: Depletion of trabecular meshwork cell numbers is a feature of the outflow system in aging and in primary open-angle glaucoma. It is possible that migration stimulated by factors present in aqueous humor may contribute to the cell loss. This investigation assessed the chemoattractant potential of glaucomatous and nonglaucomatous human aqueous humor and fibronectin, one of its constituents, on a range of cultured trabecular meshwork cell lines. METHODS: Migration was assessed in 48-well modified Boyden chambers. The potential migratory stimulants were soluble fibronectin and glaucomatous and nonglaucomatous aqueous humor. The glaucomatous aqueous samples were collected from patients undergoing trabeculotomy for primary open-angle glaucoma and the normal aqueous from normal bovine eyes and patients undergoing cataract surgery. The target cell types were normal human and bovine meshwork cells grown from explants and two human transformed meshwork cell lines from a normal (HTM-5) and a glaucomatous (HTM-3) source. RESULTS: Soluble fibronectin stimulated all the target cells to migrate with an optimal concentration ranging from 1 to 30 microg/ml, and Zigmond Hirsch checkerboard analysis indicated that both chemotaxis and chemokinesis took place. All the aqueous humor samples stimulated migration of the meshwork cell lines at an optimal concentration of 200 microl/ml. Glaucomatous aqueous humor stimulated a greater migratory response than nonglaucomatous aqueous for two of the four target cell types (P < or = 0.03). Neutralization of the fibronectin content of nonglaucomatous and glaucomatous aqueous by addition of excess anti-fibronectin antibody indicated that fibronectin could account for 35% to 80% of the migratory activity of the aqueous. CONCLUSIONS: Aqueous humor contains potentially powerful chemoattractants for trabecular meshwork cells. The activity of one of these constituents, fibronectin, has been accounted for by this study. Glaucomatous aqueous appears to be as good and in some cases a better migratory stimulant than nonglaucomatous aqueous in vitro. The migratory evidence points to a trend that may help to explain cell loss in the aging meshwork and possibly some of the extra loss in primary open-angle glaucoma.     

人眼小梁细胞体外培养及生物学特性研究

目的:建立人眼小梁细胞培养方法并研究其生物学特性。方法:以体外组织块培养的方法获得培养的人小梁细胞,应用光镜、电镜观察细胞的形态学特征,并观察其免疫组化特性和细胞的生长曲线。结果:光镜下小梁细胞为扁平多角形、单层生长;电镜下细胞连接为点粘连和缝隙连接、细胞表面可见微绒毛、胞浆细胞器丰富;免疫组化染色对抗纤维连接蛋白(Anti—FN)、抗层粘连蛋白(Anti—LN)、抗神经元特异性烯醇化酶(Anti—NSE)单抗呈阳性,对抗第Ⅷ因子(Anti-ⅧFactor)单抗呈阴性;传代小梁细胞繁殖时间较长,10天后为平台期。结论:根据体外培养的小梁细胞的形态特点、生长特征、免疫组化特性可对其进行鉴定。人小梁细胞体外培养的成功,为在细胞和分子水平研究青光眼的发病机制提供了有利的条件。眼科学报 1996;12:64—69。     

Isolation of primary open-angle glaucomatous trabecular meshwork cells from whole eye tissue

PURPOSE: Isolation and culture of human trabecular meshwork (TM) cells from primary open-angle glaucomatous (POAG) tissue has proven difficult. The objective of this study was to directly compare the utility of two different isolation methods to obtain viable human TM cells from POAG whole eye tissue. METHODS: Using a blunt dissection technique, human TM tissue was obtained from four pairs of donor eyes (67, 77, 81 and 82 years) with a documented history of POAG. TM tissue from one eye was explanted into tissue culture. TM from the contralateral eye was digested with a collagenase mixture and seeded onto culture plates. RESULTS: Primary cell isolates were obtained from all donors with both techniques. However, only cells obtained using the digestion method (3 of 4 TMs) could be passaged for expansion and freeze-downs (3 x 107 second passage cells/donor). None of the cells obtained from explanted TMs could be passaged. Cells from successful isolations were of uniform size, possessed typical TM morphology and had doubling times < 48 hours. CONCLUSION: These results demonstrate a clear advantage to digesting the extracellular matrix of glaucomatous TM tissue to obtain sufficient numbers of healthy cells for use in experiments. In contrast to cells obtained from explants, cells liberated from POAG TM tissue by digestion appear indistinguishable morphologically and behaviorally from "normal" TM cells.     

Culture of human trabecular meshwork cells and the cell characteristics of immunohistochemical studies

OBJECTIVE: To look for better cultural methods in order to obtain numerous human trabecular cells in vitro for glaucoma experimental studies, and describe the immunohistochemical characteristics of the cells. METHOD: Human trabecular meshwork cells were cultured, then 4 monoclonal antibodies were used for immunohistochemical stains of the cultured cells. RESULTS: At the primary period, the growth of human trabecular cells was obviously slower than that of cows and pigs. The immunohistochemical stains showed that the cells presented positive reactions to neuronal specific enolase (NSE) and vimentin and negative reactions to factor VIII related antigen (VIIIR:Ag) and desmin. CONCLUSIONS: The culture of human trabecular meshwork cells in vitro needs more careful and better cultural conditions. The cells originally are derived from embryonic neural crest, not from mesodermal endothelium of blood vessels. There is middle filament vimentin and no desmin in the cells.     

Preliminary observations on human trabecular meshwork cells in vitro

This report presents our preliminary observations on the trabecular meshwork from human eyes up to 5 days post-mortem in tissue culture. Satisfactory primary cultures were obtained from about 20% of the 423 explants which were investigated. The period prior to growth was from 4 days to 4 weeks and from the appearance of the initial outgrowth it took 25 to 30 days to reach maximum cellular spread within the culture chambers. The progress of the explant and the spreading of the trabecular meshwork cells was monitored by phase-contrast microscopy, time-lapse cinephotomicrography, light microscopy, transmission electron microscopy, scanning electron microscopy and autoradiography (using tritiated thymidine). On the basis of their ultrastructural appearance the cultured meshwork cells seemed to be metabolically active. Their cytoplasm contained abundant rough endoplasmic reticulum, many mitochondria, a well developed Golgi apparatus and many coated and uncoated micropinosomes. However even in short-term culture the trabecular meshwork cells had adapted to the artificial environment of our system and no longer resembled normal trabecular meshwork cells as seen in vivo. Since trabecular meshwork cells can quickly adapt their morphology in a culture environment and because the adult human meshwork contains a significant population of non-trabecular cells, the value of long term culture as a means of investigating the cellular activity of the normal and glaucomatous outflow system must be open to question.This paper was presented in part at the 7th Annual Meeting of the European Club for Ophthalmic Fine Structure in Ystad, Sweden on April 20 and 21, 1979     

The Effect of Dexamethasone on Integrin and Laminin Expression in Cultured Human Trabecular Meshwork Cells

Glucocorticoid treatment in vivo can produce a glaucoma similar in many ways to POAG. Treatment of trabecular meshwork cells in culture with dexamethasone allows the study of biochemical aspects of this disease process. The effects of dexamethasone on the expression of integrins and laminin in both normal and glaucomatous cultured human trabecular meshwork cells were evaluated. Human trabecular meshwork cell lines were cultured for 18 days in the presence or absence of 10⁻⁷mdexamethasone. Radioimmunoprecipitation was used to determine the relative expression of five αintegrin subunits. Laminin expression was evaluated with Western blots. Laminin was increased in all cell lines following dexamethasone treatment. α2, α5 and αV integrin chains showed consistent dexamethasone-induced changes in expression, while α3 and α4 subunits did not. There were no differences in the expression patterns for any of these integrin subunits between normal and glaucomatous cell lines. Increased laminin deposition as seen in this study with dexamethasone treatment may be partially responsible for the decreased outflow facility seen in both steroid-induced glaucoma and in POAG.     

人眼小梁细胞体外滤膜培养及其通透阻力测定

目的:建立人眼小梁细胞体外滤膜培养体系及通透阻力测定的模型。方法:将永生化的人眼小梁细胞株用酶消化法传代于细胞培养池的PET膜上,观察细胞的生长情况,同时分别在细胞融合后3、5d和1、2周,应用内皮细胞电阻测量仪(EVOM)测定滤膜上单层小梁细胞电阻,客观评价其通透阻力。结果:人眼小梁细胞在滤膜上生长良好,单层融合后3、5d和1、2周的平均电阻分别为(36.4±1.4)Ω、(35.0±1.7)Ω、(36.1±2.9)Ω、(39.3±3.0)Ω,各时间点无显著统计学差异(P=0.305)。结论:人眼小梁细胞滤膜上培养单层融合后,在不同时间段其阻力基本稳定。滤膜培养体系及应用EVOM对其通透阻力的测定可作为今后对小梁细胞特性研究的一个新的较好模型。     

Presence of an established calcification marker in trabecular meshwork tissue of glaucoma donors

PURPOSE: To determine the presence of calcification markers in the trabecular meshwork tissue from glaucoma donors and in trabecular meshwork cells insulted by dexamethasone (DEX) and transforming growth factor beta2 (TGFbeta2), factors associated with glaucoma. To investigate as well the effect of silencing the inhibitor of calcification matrix Gla (MGP) in the trabecular meshwork cells. METHODS: Trabecular meshwork tissue was obtained from perfused postmortem anterior segments of glaucomatous and normal eyes. Primary trabecular meshwork cells were obtained from residual corneal rims after surgical corneal transplantation. Calcification marker alkaline phosphatase (ALP) enzyme activity was assayed by fluorescence produced after substrate cleavage. DNA quantification was evaluated by fluorescence produced after binding to the Hoechst dye. Transfection of siRNA to primary cells was accomplished by nucleofector electroporation with trabecular meshwork-optimized conditions. cDNA quantification was performed with the use of TaqMan real-time PCR. RESULTS: Human trabecular meshworks from glaucoma donors exhibited significantly higher levels of ALP activity than their matched counterparts with normal eyes. The normalized ALP of the control specimens was 7.3 +/- 1.6 ng ALP/microg DNA (n = 4), whereas that of the glaucomatous tissue was 37.0 +/- 10.7 ng ALP/microg genomic DNA (n = 5; P 相似文献   

原发性开角型青光眼小梁细胞的体外培养及其生物学特性

背景原发性开角型青光眼（POAG）是一种常见致盲性眼病,其特点是房水外流阻力增加导致眼压增高。位于房水外流通道的小梁网调节房水的外流,因此研究小梁网细胞的生物学特性有着重要的意义。目的探讨POAG小梁细胞体外培养的方法及其生物学特性。方法经小梁切除术收集8例开角型青光眼患者患眼的带小梁网的深层巩膜组织块进行体外原代和传代培养,用鼠抗人层黏连蛋白（LM）单克隆抗体、兔抗人纤维连结蛋白（FN）单克隆抗体、鼠抗人神经元特异性烯醇化酶（NSE）单克隆抗体进行免疫组织化学检测以对传代细胞进行鉴定,在透射电子显微镜下对传代细胞的超微结构进行观察,并将传代小梁细胞的生物学特性与本研究组前期培养的正常小梁细胞进行比较。结果组织块培养10d左右,可见细胞从其边缘向外生长。传代细胞在4d内处于对数生长期,其后进入平台期,第7天细胞基本融合。第3代POAG小梁细胞及正常人眼小梁细胞中可见FN、LM和NSE均呈阳性表达,证实传代细胞为小梁细胞,而空白对照组细胞未见FN、LM和NSE表达。第3代POAG小梁细胞和正常小梁细胞中FN的A450值分别为0．354±0．06和0．26±0．01,LM的A450值分别为0．34±0．03和0．25±0．02,差异均有统计学意义（FN：t=14．446,P=0．001;LM：t=9．346,P=0．001）。与正常小梁细胞比较,第3代POAG小梁细胞表面的微绒毛、细胞质的溶酶体及吞噬小泡含量减少。结论采用组织块培养法可成功在体外培养POAG小梁细胞,该研究结果为研究青光眼的发病机制提供了细胞学基础。     

The myocilin (MYOC) gene expression in the human trabecular meshwork

PURPOSE: We previously reported a novel cytoskeletal protein with a myosin-like domain which is localized in the ciliary rootlet and basal body of connecting cilium of photoreceptor and hence we named it 'myocilin'. It was soon realized that myocilin is identical to a protein called TIGR (trabecular meshwork inducible glucocorticoid response protein) which was found to be responsible for the pathogenesis of juvenile open angle glaucoma. In this study, we employed in situ RNA hybridization to examine the myocilin (MYOC)/ TIGR gene expression in the trabecular meshworks of glaucomatous and nonglaucomatous eyes. METHODS: The glaucomatous specimens were obtained by trabeculectomy from the patients with primary open angle glaucoma (POAG), chronic angle closure glaucoma (CACG) and steroid glaucoma, respectively, and the nonglaucomatous specimens were obtained from a victim of traffic accident at autopsy and from a patient with maxillary sinus carcinoma at enucleation for the operation. The in situ RNA hybridization was carried out with digoxigenin-labeled sense and antisense RNA probes. RESULTS: In all cases, hybridization signals were detected primarily in the trabecular meshwork cells and secondarily in the fibroblast-like cells of corneoscleral wall. CONCLUSIONS: Myocilin gene is expressed clearly in the trabecular meshwork cells of both glaucomatous and nonglaucomatous eyes.     

Sialyl Lewis X, Lewis X, and N-acetyllactosamine expression on normal and glaucomatous eyes

PURPOSE: Sialyl Lewis X (sLex), Lewis X (Lex), and N-acetyllactosamine are carbohydrate chains of neolactoglycoconjugates which are expressed by specific cell types and are important in the functioning of cells within an organism. This study attempts to determine the expression of these glycoconjugates on the conjunctiva, cornea, and trabecular meshwork (TM) of both normal and glaucomatous eyes. METHODS: Frozen anterior segment sections of both normal and glaucomatous human cadaver eyes, as well as rabbit eyes, were stained with a panel of monoclonal antibodies (mAbs) to neolactoglycoconjugates using an Avidin Biotin Peroxidase Complex/Alkaline Phosphatase staining method. RESULTS: SLex characteristically stained both human conjunctival and corneal epithelia in normal (n=5) and glaucomatous (n=5) sections. SLex stained corneal and conjunctival epithelia of glaucomatous eyes much more intensely than normal eyes. Rabbit cornea sections stained for sLex, Lex, and N-acetyllactosamine. However, human cornea only consistently stained with sLex. Normal and glaucomatous human TM sections did not stain for sLex, Lex, or N-acetyllactosamine. CONCLUSIONS: The expression of glycoconjugates with sLex side chains appears to be upregulated in the conjunctival and corneal epithelia of glaucomatous eyes. Distinct species specific differences were noted in Lex and N-acetyllactosamine staining patterns in rabbit and human corneal epithelia.     

Fibronectin in human trabecular drainage channels

Fibronectin, an extracellular glycoprotein, has been shown to be produced by human trabecular cells in culture by our group as well as Polansky and co-investigators. Studies of Rodrigues et al suggested that fibronectin may be one of several glycoproteins found in increased amounts in the corneoscleral trabecular meshwork of glaucomatous eyes. The authors have developed a sensitive immunoassay utilizing avidin-biotinylated enzyme complex (ABC) to detect low levels of fibronectin in frozen sections of human eyes. The authors have used this immunoassay together with a perfusion technique to demonstrate distribution patterns of fibronectin present in human aqueous drainage channels. The authors found that fibronectin is present in larger quantities in the aqueous drainage channels than in the surrounding tissues in 18 eyes from older patients.     

Carbohydrate binding proteins galectin-1 and galectin-3 in human trabecular meshwork

PURPOSE: Galectins are a family of carbohydrate binding proteins involved in a variety of biological processes including cell-cell and cell-matrix interactions. We examined galectin-1 and galectin-3 to determine if galectins are expressed in the human trabecular meshwork and Schlemm's canal. METHODS: Human trabecular meshworks were dissected from donor eyes within 12 hr of death. Galectin-1 and galectin-3 expression was examined by RT-PCR and Western blot analysis. Immunohistochemistry of galectin-1 and galectin-3 was analyzed in normal and glaucomatous tissue. RESULTS: Expression of mRNA and protein of Galectin-1 (14 kDa) and galectin-3 (31 kDa) was found in the outflow pathway. Immunostaining revealed galectin-1 and galectin-3 throughout the meshwork, cells lining Schlemm's canal, and extracellular spaces in the inner and outer walls of the canal. Comparison of normal, POAG and PEX samples revealed no difference in location or intensity for either galectin-1 and galectin-3. CONCLUSION: Galectin-1 and galectin-3 are present in human trabecular meshwork of normal and glaucomatous eyes.     

The effect of epinephrine and benzalkonium chloride on cultured corneal endothelial and trabecular meshwork cells

We evaluated the effect of dipivefrin hydrochloride, epinephrine hydrochloride, epinephrine borate and their respective vehicles with and without the preservative benzalkonium chloride, on the in vitro growth characteristics of human corneal keratocytes, endothelial cells and trabecular meshwork. Epinephrine hydrochloride and borate at low concentrations (0.0002%) significantly inhibited growth of both trabecular meshwork and corneal endothelial cells. Higher concentrations (0.02%) of these same drugs induced the same effect on the growth of keratocytes in vitro. Similar observations were made on the effect of dipivefrin hydrochloride on human corneal cells in vitro. Benzalkonium chloride alone was demonstrated to be responsible for the growth inhibitory effects on trabecular cells. The susceptibility of trabecular meshwork cells in culture to the commonly used ophthalmic preservative benzalkonium chloride is demonstrated.     

地塞米松诱导人小梁细胞bcl—2基因的表达

目的:探讨地塞米松对小梁细胞bcl-2基因表达的影响。方法:应用组织块培养的方法建立人小梁细胞培养。取第三代小梁细胞培养于载玻片上,含10~(-7)mol/L地塞米松的培养液作用6小时、12小时、24小时后,应用LSAB观察bcl-2基因表达的情况。结果:地塞米松作用6小时后可见bcl-2蛋白阳性细胞,且随时间的延长,阳性细胞有所增多。结论:地塞米松可诱导小梁细胞bcl-2基因表达,可能在激素性青光眼发病中起着一定的作用。眼科学报1998;14:187～189。     

Human trabecular meshwork phagocytosis. Observations in an organ culture system

Perfusion organ culture of the trabecular meshwork was used to study the phagocytic ability of human trabecular cells. Cultured eyes were challenged with blood, latex microspheres, or zymosan particles for periods of 1 hour to 7 days. Trabecular cells were capable of ingesting all three types of particles. The presence of a foreign particle did not necessarily induce a phagocytic response, however, as free particles were seen in the intertrabecular spaces and Schlemm's canal. In contrast to studies in animals which indicate trabecular cell migration from the eye may play a role in the removal of foreign debris, limited human trabecular cell migration was observed. The effect of the culture process on trabecular cell phagocytosis was also studied, using adult cats. One eye received a phagocytic challenge in vivo with the fellow eye later receiving the phagocytic challenge in vitro. Phagocytosis was demonstrated in each eye, although more cells were involved with phagocytosis in vivo. The additional cells involved in vivo may have been recruited by an accompanying inflammation. Organ culture of human trabecular meshwork may be useful in the study of trabecular cell phagocytosis, and it allows separation of the effects of inflammation from the potential effects of phagocytosis itself.