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。     

Clinical,structural and molecular phototherapy effects of laser irradiation on the trabecular meshwork of human glaucomatous eyes

The effects of argon laser trabeculoplasty (LTP) on intraocular pressure (IOP), outflow facility, the morphology of the trabecular meshwork (TM), and the pattern of extracellular glycoprotein fibronectin in trabeculum were studied in 46 eyes of patients with primary open-angle glaucoma (POAG). The LTP was done with informed consent, anticipating that trabeculectomy would be carried out at a scheduled time (2 h to several months following laser therapy). We found that the magnitude of IOP reduction and the improvement in the facility of outflow achieved are directly dependent on the time course after LTP and laser-induced structural changes in trabecular tissue. Light microscopic and immunohistochemical evaluations of the TM specimens at earlier intervals after LTP revealed evidence of heat effects, with disruption and shrinkage of the TM collagenous components and accumulation of fibronectin deposits in the aqueous drainage channels as compared with the TMs of matched patients with POAG who did not receive laser treatment. Within 24 h after LTP, proteins of glaucomatous TMs excised from patients incorporated increased amounts of [³H]-leucine radioactive label; however, the amount of [³H]-leucine-labeled material was significantly depressed in later periods of evaluation. The specimens obtained at longer intervals after LTP showed partial or total occlusion of the intertrabecular spaces by extracellular debris; however, the amount of trabecular fibronectin was not significantly different from that measured 24 h after LTP. At least two potential mechanisms are proposed for the TM tissue response to laser treatment, including heat-induced stretching of the collagen in lamellae and fibronectin-mediated attachment of beams supporting an adhesive tightening of the trabecular components caused by LTP. The changes in laser-induced tissue responses appear to be the result of morphological repair of irradiation-injured trabecular tissue.     

Effects of TGF-beta2 in perfused human eyes

PURPOSE: TGF-beta2 is known to be present at elevated levels in the aqueous humor of patients with primary open-angle glaucoma (POAG). Studies have shown that TGF-beta2 influences cultured trabecular meshwork (TM) cells, but the effects of this cytokine on intact TM and outflow facility have not been studied. The purpose of this study was to investigate whether TGF-beta2 treatment induces changes in outflow facility and morphologic changes in the TM tissue and whether these changes are comparable to those previously recorded in glaucomatous eyes. METHODS: Baseline facility was measured in paired human eyes (n = 8 pairs), with a constant-flow anterior segment culture system. Medium perfusing experimental eyes was then supplemented with activated human recombinant TGF-beta2 (3.0 ng/mL, comparable to or slightly greater than measured aqueous humor levels in patients with POAG), and facility was measured for at least 8 days. At the conclusion of the perfusion, eyes were fixed and processed for light microscopy, transmission electron microscopy, and immunolabeling studies. RESULTS: TGF-beta2 perfusion reduced outflow facility by 27% (P = 0.03) and promoted focal accumulation of fine fibrillar extracellular material in multilayered structures under the inner wall of Schlemm's canal. In treated eyes, Schlemm's canal was 27% shorter (P = 0.02), and the length of the inner wall apparently available for fluid flow was 33% less (P = 0.001), both compared with paired control eyes. CONCLUSIONS: TGF-beta2 reduces outflow facility when perfused into cultured human anterior segments. Furthermore, TGF-beta2 affects the extracellular matrix of the trabecular meshwork in a manner that is consistent with the observed reduction in outflow facility. Although the distribution of accumulated fibrillar material was different in these perfused eyes than that in POAG, the difference could be due to variation in biomechanical environment for TM cells in cultured anterior segments compared with the living eye. Overall, these results support the hypothesis that elevated TGF-beta2 levels in the aqueous humor play a role in the pathogenesis of the ocular hypertension in POAG.     

Glycans of the trabecular meshwork in primary open angle glaucoma.

AIMS: Glycan expression was compared in glaucomatous trabecular meshwork (TM) and normal TM in order to determine any differences which may reflect pathological changes underlying primary open angle glaucoma (POAG). METHODS: Resin embedded TM from trabeculectomy specimens from 15 eyes with POAG and from 12 eyes with normal anterior segments were probed with a panel of biotinylated lectins and an avidin-peroxidase revealing system at the light microscope level. Statistical analyses were performed on the comparative staining results. RESULTS: The lectins ConA and ePHA showed strong staining in all areas of both glaucomatous and normal TM; ePHA staining of Schlemm's canal (SC) from POAG TM was significantly less than that from normal TM (ePHA-SC p = 0.04). The lectins PSA, LCA, and SNA bound moderately strongly to SC endothelium and weakly to the endothelium of the corneoscleral meshwork (CSM); glaucomatous SC endothelial binding was significantly less than that of normal SC endothelium for PSA and LCA (PSA-SC p = 0.002, LCA-SC p = 0.002). STA and DSA showed moderately strong binding while WGA, ECA, AHA, and MPA bound weakly throughout the TM; for DSA and MPA this staining was significantly greater in POAG than in normal TM (DSA-SC p = 0.001, DSA-CSM p = 0.002, MPA-SC p = 0.01, MPA-CSM p = 0.02). Jac stained strongly throughout the TM and showed no significant difference in POAG compared with normal TM (Jac-SC p = 0.6, Jac-CSM p = 1). 1PHA, SBA, DBA, CTA, UEA-1 and LTA did not bind to glaucomatous TM or normal TM. There were no age-related changes seen. CONCLUSIONS: The expression of some complex and hybrid, bisected and non-bisected N-linked glycans is significantly diminished in glaucomatous TM compared with normal TM. Some glycans with multiple N-acetylglucosamine residues and O-linked glycans with terminal and subterminal galactosyl groups are significantly increased in POAG TM. Glycan expression does not change significantly with age in POAG or normal TM.     

The matricellular protein SPARC is expressed in human trabecular meshwork

PURPOSE: This investigation was undertaken to determine whether the matricellular protein SPARC is expressed in the human trabecular meshwork (TM) and cultured human trabecular meshwork cells. METHODS: Human donor trabecular meshwork and cultured cells obtained from trabecular meshwork were used in this study. Total RNA was obtained from TM and cultured TM endothelial cells, and RT-PCR was done with primers specific for SPARC. Western blotting was performed on donor TMs using an anti-SPARC monoclonal antibody prepared against rHuSPARC. Confocal microscopy was used to determine the distribution of SPARC in human anterior segments, and immunofluorescence on cultured TM cells was performed with the anti-SPARC antibody. RESULTS: SPARC mRNA was expressed both in TM and in cultured TM cells. Immunoblotting for SPARC showed a doublet with a molecular mass approximately 43 kDa. The ratio of the doublet bands varied with each of the samples; some of the cultured cells and the tissue samples exhibited more of the upper band, and other cultured cells contained almost equal amounts of the two bands. The upper band was shown to be a glycosylated form of SPARC. Immunofluorescence showed that SPARC was expressed in the cultured TM, and confocal microscopy with the anti-SPARC antibody demonstrated the presence of this protein in the TM and in other tissues in the anterior segment. CONCLUSIONS: Our data conclusively show that SPARC mRNA and protein are present in non-glaucomatous TM tissue and in cultured TM cells. Because of its effect on matrix metalloproteinases, SPARC may play a role in the regulation of intraocular pressure.     

Regulation of glucocorticoid responsiveness in glaucomatous trabecular meshwork cells by glucocorticoid receptor-beta

PURPOSE: Glucocorticoid administration can lead to increased intraocular pressure in greater than 90% of patients with primary open-angle glaucoma (POAG), compared with 30% to 40% of the general population. The molecular mechanisms for increased steroid responsiveness among patients with glaucoma are unknown. An alternative splicing variant of the human glucocorticoid receptor GRbeta has dominant negative activity and has been implicated in a variety of steroid-resistant diseases. GRbeta also may play a role in glucocorticoid hyperresponsiveness in glaucoma. METHODS: Western blot analysis was performed to detect the expression of GRalpha and GRbeta in TM cells and its regulation by dexamethasone (DEX). Immunocytochemistry was used to compare the subcellular expression of GRbeta between normal and glaucomatous TM cell lines. DEX transgene induction in a luciferase reporter was performed to investigate the differential glucocorticoid responsiveness between multiple normal and glaucomatous TM cell lines. Overexpression of GRbeta was conducted in glaucomatous TM cell lines, and the regulation of GRbeta in the Dex-induced reporter gene luciferase or endogenous myocilin and fibronectin expression were determined. RESULTS: Trabecular meshwork (TM) cell lines derived from normal individuals expressed higher levels of GRbeta than did glaucomatous TM cells. Glaucomatous TM cells were more susceptible to DEX induction of a luciferase reporter gene than were TM cells derived from normal donors. Overexpression of GRbeta in glaucomatous TM cells inhibited DEX induction of a luciferase reporter gene as well as the endogenous genes MYOC and fibronectin. CONCLUSIONS: The decreased amount of GRbeta in glaucomatous TM cells could result in enhanced glucocorticoid responsiveness and ocular hypertension.     

The Study on Culture and Ultrastructure of Glaucomatous Trabecular Cells in Vitro

Purpose : To establish the culture system of human glaucomatous trabecular cells in vitro and study their ultrastructures.Methods : The trabecular specimens from trabeculectomy were cultured in vitro and passaged 3 times, then identified. Moreover, the glaucomatous cells were observed with electron microscope while compared with the normal ones.Result: Cultured human glaucomatous trabecular cells were obtained. The ultrastructure of the cells showed the decrease in vilious project, coated vesicle and lysosomal inclusion. Conclusion : The establishment of human glaucomatous trabecular cells culture in vitro made the culture system more perfect. The morphologic changes might be related to the abnormal functions of human trabecular meshwork cells. Eye Science 1998; 14 : 134 - 737.     

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     

Evaluation of electrofunctional data following argon-laser trabeculoplasty in primary open-angle glaucoma

To establish whether or not glaucomatous damage is reversible, we obtained pattern-reversal electroretinograms (PERGs) and visual evoked potentials (VEPs) in 25 eyes of 25 patients suffering from bilateral primary open-angle glaucoma (POAG) before and after argon-laser trabeculoplasty. The laser treatment was carried out in only one eye chosen at random, and the fellow eye was used as a control. In the present study we intended to verify the possibility of using electrofunctional techniques to determine the two distinct and, probably, consecutive glaucomatous alterations occurring in ganglion cells: functional (reversible) and anatomical (irreversible). The results obtained indicate that glaucomatous damage is irreversible. We propose that such alterations differ very slightly and that the current electrofunctional techniques may not be sufficiently sophisticated to distinguish between them.     

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     

正常人和原发性开角型青光眼患者小梁网M3受体的表达

目的 研究正常人和晚期原发性开角型青光眼（primary open angle glaucoma,POAG）患者小梁网M3受体的表达,探讨POAG患者小梁网的病理生理改变。方法 取5例正常人及10例晚期POAG患者小梁网组织标本,应用免疫组化方法检测小梁细胞M3受体,并利用计算机图像分析系统对检测结果进行分析。结果 5例正常人的小梁网组织标本的M3受体表达,阳性细胞主要分布在葡萄膜小梁网处,前至Schwalbe线,后达巩膜突。晚期POAG患者小梁网组织标本中,小梁细胞数量明显减少,M3受体阳性细胞亦相应减少且散在分布,部分标本甚至无阳性M3受体表达。结论 正常人小梁网细胞M3受体表达阳性,晚期POAG患者小梁网M3受体阳性细胞数量明显减少且分布不规则。     

Donor corneoscleral buttons: a new source of trabecular meshwork for research

The human trabecular meshwork (TM) is the major site of resistance for aqueous humor outflow. The purpose of this investigation was to determine the suitability of TMs harvested from donor corneoscleral buttons (buttons) for laboratory studies.Histologic examination using light and electron microscopy was performed. Additionally, the effect of incubation in serum free media on histologic appearance was studied. Total RNA extraction was performed on 45 buttons and four whole eyes. Some TMs were used as a source for establishing primary cell cultures of TM endothelial cells.Compared with donor whole eyes, light microscopy showed comparable TM cellularity in the juxtacanalicular and corneoscleral TM; there was some loss of cells in the inner portion of uveal TM that was adjacent to the anterior chamber. The TM cells appeared healthy and structurally normal on transmissive electron microscopy. Usable quantities of total RNA could be extracted from buttons that had been stored up to 5 weeks. The amount of total RNA extracted correlated well with the histologic appearance. Incubation in serum free media did not have an effect on the histologic appearance. All attempts at establishing primary cell cultures were successful.Unused, donor corneoscleral buttons are an excellent source of TM.     

Expression of nitrotyrosine and oxidative consequences in the trabecular meshwork of patients with primary open-angle glaucoma

PURPOSE: To evaluate the possible correlation between the visual field defects in patients with primary open-angle glaucoma (POAG) and the expression and enzymatic activity of nitric oxide synthase (NOS) isoenzymes and nitrotyrosine in trabecular meshwork (TM) samples. METHODS: TM specimens were collected from 146 patients with POAG by using standard filtration surgery. Visual field defects were evaluated by perimetry. Expression of endothelial (e)NOS and inducible (i)NOS were evaluated by quantitative RT-PCR. Constitutive (Ca2+-dependent) and iNOS (Ca2+-independent) activities were measured by the conversion of L-[14C]-arginine to L-[14C]-citrulline. In four TM specimens from POAG-affected eyes and in three human donor control eyes, 3-nitrotyrosine was localized by immunohistochemistry. The marker of lipid peroxidation malondialdehyde (MDA) was measured by the thiobarbituric acid test in samples of aqueous humor (AH) from 48 patients with either POAG or cataracts. RESULTS: The results showed an upregulation of iNOS and a downregulation of calcium-dependent NOS correlated with visual field defects. Expression and activity of iNOS increased in parallel with visual field defects. However, constitutive activity decreased as the visual field defect increased. Nitrotyrosine was observed only in the cells of the TM specimens from eyes with severe POAG. CONCLUSIONS: The increased expression and activity of iNOS in the TM of patients with POAG are proportional to the visual field defect and could lead to the increased of nitrotyrosine levels which may serve as marker of oxidative stress in the progression of cell death of the TM in POAG.     

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.     

Cell culture of the human lamina cribrosa

The extracellular matrix of the lamina cribrosa may be important in the changes in the optic nerve head associated with glaucoma. To investigate the cell biology of this tissue, human lamina cribrosa was explanted in tissue culture and two cell types grown from this tissue were characterized. The most common cell type obtained was a large, flat, polygonal cell which was negative for glial fibrillar acidic protein (GFAP) and could be serially subcultured. This cell type synthesized collagens type III and type IV, fibronectin and elastin. Much less commonly grown was a cell type with conspicuous long processes and which was positive for GFAP. This presumed astrocyte synthesized collagen type IV and fibronectin. Fibroblastic cells were not obtained from this tissue but were easily grown from sclera. The cells that we have cultured from the human lamina cribrosa may produce the extracellular matrix present in the cribriform plates of this tissue and be important in the glaucomatous process.     

Glaucoma in a rural population of southern India: the Aravind comprehensive eye survey

PURPOSE: To determine the prevalence of glaucoma and risk factors for primary open-angle glaucoma in a rural population of southern India. DESIGN: A population-based cross-sectional study. PARTICIPANTS: A total of 5150 subjects aged 40 years and older from 50 clusters representative of three southern districts of Tamil Nadu in southern India. METHODS: All participants had a comprehensive eye examination at the base hospital, including visual acuity using logarithm of the minimum angle of resolution illiterate E charts and refraction, slit-lamp biomicroscopy, gonioscopy, applanation tonometry, dilated fundus examinations, and automated central 24-2 full-threshold perimetry. MAIN OUTCOME MEASURES: Definite primary open-angle glaucoma (POAG) was defined as angles open on gonioscopy and glaucomatous optic disc changes with matching visual field defects, whereas ocular hypertension was defined as intraocular pressure (IOP) greater than 21 mmHg without glaucomatous optic disc damage and visual field defects in the presence of an open angle. Manifest primary angle-closure glaucoma (PACG) was defined as glaucomatous optic disc damage or glaucomatous visual field defects with the anterior chamber angle partly or totally closed, appositional angle closure or synechiae in the angle, and absence of signs of secondary angle closure. Secondary glaucoma was defined as glaucomatous optic nerve damage and/or visual field abnormalities suggestive of glaucoma with ocular disorders that contribute to a secondary elevation in IOP. RESULTS: The prevalence (95% confidence interval) of any glaucoma was 2.6% (2.2, 3.0), of POAG it was 1.7% (1.3, 2.1), and if PACG it was 0.5% (0.3, 0.7), and secondary glaucoma excluding pseudoexfoliation was 0.3% (0.2,0.5). On multivariate analysis, increasing age, male gender, myopia greater than 1 diopter, and pseudoexfoliation were significantly associated with POAG. After best correction, 18 persons (20.9%) with POAG were blind in either eye because of glaucoma, including 6 who were bilaterally blind and an additional 12 persons with unilateral blindness because of glaucomatous optic neuropathy in that eye. Of those identified with POAG, 93.0% had not been previously diagnosed with POAG. CONCLUSIONS: The prevalence of glaucoma in this population is not lower than that reported for white populations elsewhere. A large proportion of those with POAG had not been previously diagnosed. One fifth of those with POAG had blindness in one or both eyes from glaucoma. Early detection of glaucoma in this population will reduce the burden of blindness in India.     

Increased expression of serum amyloid A in glaucoma and its effect on intraocular pressure

PURPOSE: To search for and validate potential molecular pathogenic mechanisms in the trabecular meshwork (TM) responsible for the elevated intraocular pressure (IOP) associated with glaucoma. METHODS: Gene chip arrays were used to identify differential gene expression in glaucomatous TM tissues. Serum amyloid A (SAA) upregulation was subsequently confirmed with quantitative PCR (QPCR) and ELISA. The effect of SAA on gene expression of cultured human TM cells was tested with gene chip arrays and verified with ELISA, and its effect on IOP was evaluated in the human ocular perfusion organ culture. RESULTS: Microarray analysis showed that the expression of SAA2 was increased in TM tissues from donors with glaucoma. This finding was subsequently confirmed by QPCR. The SAA mRNA levels were increased in glaucoma TM tissues by more than 5-fold (P < 0.05) and in cultured TM cells derived from donors with glaucoma by 25-fold (P < 0.05) compared with controls. SAA protein levels in the TM of glaucoma patients were also significantly (P < 0.05) elevated by 2.9-fold. Treatment of cultured human TM cells with recombinant SAA affected gene expression, including a 22-fold up-regulation of interleukin-8 (P < 0.001). SAA increased IOP by approximately 40% (P < 0.05) in the human ocular perfusion organ culture without any observable changes in the morphology of the tissues involved in aqueous outflow. CONCLUSIONS: These findings indicate that SAA, which is an acute-phase apolipoprotein that plays important roles in infection, inflammation, and tissue repair, may contribute to the pathogenic changes to the TM in glaucoma.     

Detection of differentially expressed glycogenes in trabecular meshwork of eyes with primary open-angle glaucoma

PURPOSE: To identify differentially expressed glycogenes in trabecular meshwork (TM) of eyes with primary open-angle glaucoma (POAG). METHODS: Total RNA was isolated from TM of cadaveric eyes derived from donors with diagnosed glaucomas of different etiologies and from normal control subjects. RNA was amplified and hybridized to the GLYCOv2 oligonucleotide microarray that contains probes for carbohydrate-binding proteins, glycosyltransferases, and other genes involved in the regulation of glycosylation. Statistical analysis was used to identify differentially expressed genes between normal and POAG samples. RESULTS: This study revealed that POAG TM and normal TM have distinct gene expression profiles. Of the 2001 genes on the array, 19 genes showed differential expression of greater than 1.4-fold in POAG. Mimecan and activinA, which have been shown to be upregulated in models of glaucoma, were both found to be elevated in POAG TM. Many genes were identified for the first time to be differentially regulated in POAG. Among the upregulated genes were: (1) cell adhesion molecules including platelet endothelial cell adhesion molecule-1 and P-selectin, both of which are targets of NFkappaB, which has been shown to be activated in glaucomatous TM; (2) lumican, a core protein of keratan sulfate proteoglycans; and (3) the receptor for IL6, a cytokine that has been shown to be upregulated in TM in response to elevated intraocular pressure. Among the downregulated genes were chondroitin-4-O-sulfotransferase involved in the synthesis of chondroitin sulfate chains and the receptor for PDGFbeta, a growth factor that has been shown to stimulate both TM cell proliferation and phagocytic activity. Results for several genes were confirmed by RTq-PCR. CONCLUSIONS: Microarray technology was used to show, for the first time, that POAG TM has a distinct glycogene expression profile. Differentially expressed glycogenes identified in this study have not been previously investigated for their role in the pathogenesis of POAG and thus are novel factors for further study of the mechanism of the disease and for their possible use as diagnostic markers.     

人血管内皮细胞体外培养及鉴定

目的：探讨人血管内皮细胞培养方法,提高人血管内皮细胞培养的成功率。方法：采用“０．２％胶原酶Ⅲ灌流消化法”培养人脐静脉内皮细胞,当细胞繁殖融合后,用０．０６２５％胰蛋白酶消化法传代;根据细胞生长特点,通过形态学特征与免疫组化检查对细胞进行鉴定。结果：培养细胞２４小时完全贴壁,生长旺盛,３～５天融合传代,形态学上为典型的血管内皮细胞;鼠抗人血管皮细胞单克隆抗体（ＣＤ３４）抗体染色呈阳性。结论：“灌流