Effect of Transforming Growth Factor-β_2 on Phagocytosis in Cultured Bovine Trabecular Meshwork Cells

The effect of transforming growth factor β 2 (TGF β 2) on phagocytosis in bovine trabecular meshwork cells in vitro was investigated. After the cultured bovine trabecular meshwork cells were treated with 0 ng/ml, 0.32 ng/ml, 1 ng/ml, 3.2 ng/ml TGF β 2 for 24 h, latex beads were added into the incubation medium, and the numbers of the latex beads in 20 adjacent cells were counted under a microscope 24 h later, after treatment with Wright's stain. Our results showed that the average numbers of the latex beads in the trabecular meshwork cells treated with TGF β 2 of different concentrations were 53.1±1.7 beads/cell, 56.4±2.9 beads/cell and 77.9±6.5 beads/cell respectinvely, in comparison with 45.5±3.3 beads/cell of the control group. TGF β 2 significantly increased the number of the latex beads phagocytosed by cultured bovine trabecular meshwork cells in a dose dependent manner. TGF β 2 could promote the phagocytosis of bovine trabecular meshwork cells in vitro . It may be involved in the cellularity decrease of the trabecular meshwork in the patients of primary open angle glaucoma through promoting the phagocytosis of trabecular meshwork cells.

Existence of Heme Oxygenase-carbon Monoxide-cyclic Guanosine Monophosphate Pathway in Human Trabecular Meshwork Cells In Vitro

To confirm the existence of heme oxygenase (HO)-carbon monoxide (CO)- cyclic guanosine monophosphate (cGMP) pathway in the cultured human trabecular meshwork cells (HTMCs) in vitro, and to evaluate the inductive role of hemin on this pathway, HTMCs of the third to fourth generation were cultured in vitro. Reverse transcripase-polymerase chain reaction (RT-PCR) was employed for detection of HO-1 and HO-2 mRNA. Immunohistochemical staining was used to detect HO-1 and HO-2 proteins. Hemin was added into the culture solution. The HO-1 mRNA levels were quantified by RT-PCR. The relative amount of carbon monoxide released into the media was measured with the quantifying carbon monoxide hemoglobin (HbCO) by spectrophotometry. Radioimmunoassay was used to determine changes of cGMP in HTMCs. The results showed that cultured cells had the specific characteristics of HTMCs. Both HO-1 and HO-2 genes were expressed in HTMCs, as well as HO-1 and HO-2 proteins in HTMCs. Hemin induced HO-1 mRNA, HbCO and cGMP in a dose-dependent manner. In conclusion, HO-CO-cGMP pathway exists in the cultured HTMCs and can be induced by hemin. Pharmacological stimulation of HO-CO-cGMP pathway may constitute a novel therapeutic approach to rescuing glaucoma.

Apoptosis of Human Trabecular Meshwork Cells Induced by Transforming Growth Factor-β_2 in vitro

Whether transforming growth factor β 2 (TGF β 2) induces apoptosis of human trabecular meshwork cells was investigated in vitro . Cultured 3 5 passage human trabecular meshwork cells were treated with 0 (control), 0.32, 1, 3.2 ng/ml TGF β 2 for 48 h and divided into control group and experimental group. The apoptosis of human trabecular meshwork cells was examined by transmisson electron microscopy, TUNEL technique and flow cytometry. The results showed characteristic morphologic changes of apoptotic cells were observed under transmission electron microscopy. DNA fragmentation of human trabecular meshwork cells was found by TUNEL technique. Quantitative analysis of flow cytometry showed that percentages of apoptotic human trabecular meshwork cells were (2.79±0.44) %, (4.43 ±1.17) % and (9.60±2.05) % respectively with different concentrations [1 ng/ml ( P< 0.05), 3.2 ng/ml ( P< 0.01)] of TGF β 2 with the difference being significant between experimental group and control group[(1.41±0.34) %]. It was concluded that TGF β 2 can induce apoptosis of human trabecular meshwork cells in vitro and may be involved in the decrease of trabecular meshwork cells in the patients with primary open angle glaucoma and aging of normal people.

Inhibition of latrunculin-A on dexamethasone-induced fibronectin production in cultured human trabecular meshwork cells

AIM: To determine the effects of a low dose latrunculin (LAT)-A on dexamethasone (Dex)-induced upregulation of extracellular matrix proteins fibronectin (FN) in cultured human trabecular meshwork (HTM) cells. METHODS: HTM cells were cultured to confluent and incubated with 0.4 mu mol/L Dex and/or 0.05 mu mol/L LAT-A. FN expression in HTM cells was evaluated by Western blot and immunofluorescence microscopy. RESULTS: Dex up-regulated FN production in HTM cells, failed to do so when co-incubated with LAT-A. LAT-A decreased production of FN in cultured HTM cells. CONCLUSION: This study indicated that LAT-A may modulate the expression of fibronectin in trabecular meshwork to achieve treatment for steroids and other types of glaucoma. It has an important prospect as an intraocular pressure-lowering drug.

Antagonistic Effects of Tranilast on Proliferation and Collagen Synthesis Induced by TGF-β_2 in Cultured Human Trabecular Meshwork Cells

Whether tranilast had antagonistic effect on proliferation inhibition and collagen synthesis promotion induced by TGF-β 2 in cultured human trabecular meshwork cells was investigated. Suspension of 1×104 cultured human trabecular meshwork cells of 3—5 passage was distributed in each well of a 96-well disk and divided into control group and experimental group. After 24 h, 0 μg/ml (control), 12.5 μg/ml, 25 μg/ml, 50 μg/ml tranilast with 3.2 ng/ml TGF-β 2 were added into the incubation medium. Another 24 h later, proliferation and collagen synthesis in cultured human trabecular meshwork cells were examined respectively by using tetrazolium-based semiautomated colormetric (MTT) assay and 3H-proline incorporation with liquid scintillation technique. The results showed absorbance (A) values of the experimental groups were 0.9036±0.3017, 1.1361± 0.1352, 1.2457±0.1524 according to the different concentrations of tranilast, and 0.8956± 0.1903 of the control group. In comparison with the control group, 25 μg/ml (q'=3.23, P< 0.05), 50 μg/ml (q'=4.70, P<0.01) tranilast significantly antagonized the decrease of the A values induced by TGF-β 2 in the cultured human trabecular meshwork cells. In comparison with the control group [817.37±124.21 cpm/104 cells], 12.5 μg/ml (620.33±80.46 cpm/104 cells, q'= 4.26, P< 0.05), 25 μg/ml (594.58±88.13 cpm/104 cells, q'=4.81, P<0.01), 50 μg/ml (418.64±67.90 cpm/104 cells, q'=8.62, P<0.01) tranilast significantly inhibited the incorporation of 3H-proline into the cultured human trabecular meshwork cells promoted by TGF-β 2 in a dose-dependent manner. It was concluded that tranilast had the antagonistic effect on the proliferation inhibition and collagen synthesis promotion induced by TGF-β 2 in the cultured human trabecular meshwork cells.

Inhibitory Effect of Tissue Transglutaminase (tTG) Antisense Oligodeoxynucleotides on tTG Expression in Cultured Bovine Trabecular Meshwork Cells

To study the effect of tTG fully phosphorothioated antisense oligodeoxynucleotides （tTG-ASDON） on tTG expression in cultured bovine trabecular meshwork cells （BTMCs） in vitro and explore a new treatment alternative for primary open angle glaucoma （POAG）, the ASDON1 and ASDON2 complementary to the protein codogram region of tTG were designed, synthesized and phosphorothioated according to the secondary structure of tTG. The ASDON1 and ASDON2 were embedded in Lipofectamine and transfected into BTMCs. The untreated group served as negative controls. The expression of tTG in the mRNA and protein level were measured by semi-quantitative RT-PCR and immunohistochemical technique-Supervision method respectively. Our results showed that both the mRNA and the protein of tTG with tTG-ASDON1 and tTCr-ASDON2 were significantly decreased as compared with that of the controls （P〈0.05）. On the other hand, no significant difference was found between the ASDON1 group and the ASDON2 group. It is concluded that the expression of tTG mRNA and protein in cultured BTMC are down-regulated by tTG- ASDON. As a result, tTG-ASDON may be used for the treatment of POAG through the inhibitory effect on the expression of tTG.

Transforming Growth Factor-β_2 Gene Cloning and Protein Expression in Human Trabecular Meshwork Cells

Whether cultured human trabecular meshwork cells express transforming growth factor-β 2 (TGF-β 2) messenger RNA (mRNA) and protein was investigated. Total RNA of 10 6 cultured human trabecular meshwork cells was extracted with TRIZOL reagent, reverse transcriptase-polymerase chain reaction (RT-PCR) were used for detection of TGF-β 2 messenger RNA, and the PCR product was verified by sequencing. Immunohistochemical staining was used to detect TGF-β 2 protein. The results showed that a single RT-PCR amplified product was obtained, and the sequence was homologous to the known sequence. TGF-β 2 immunostaining was positive. It was concluded that trabecular meshwork cells could produce TGF-β 2 and contribute to the presence of TGF-β 2 in trabecular meshwork microenvironment as well as aqueous humor. Trabecular meshwork cells were affected by TGF-β 2 not only through paracrine, but also autocrine action. Whether abnormal changes in TGF-β 2 production contribute to the pathogenesis of primary open-angle glaucoma is worth further investigation.

Expression of Tissue Transglutaminase in Cultured Bovine Trabecluar Meshwork Cells

Summary: To study whether cultured bovine trabecluar meshwork cells (BTMC) are capable of expressing tTG in protein and at mRNA level, BTMC were cultured in vitro and passaged three times, then the cells were transferred onto or cultured on sterile cover or submitted to isolation of RNA with Trizol, and the expression of tTG was detected by immunohistochemical technique and reverse transcription polymerase chain reaction (RT-PCR) respectively. Our results showed that tTG immunostaining was positive in the cytoplasm and rarely in the nucleus of cultured BTMC. No immunostaining was seen in the negative control. Moreover, a single RT-PCR amplified product whose sequence and size were in accordance with our known parameters was obtained. The expression of tTG in cultured BTMC was confirmed in protein and at mRNA level. BMTC is available more readily for the investigation of the relationship between tTG and primary open-angle glaucoma.

Identification, quantification and agerelated changes of human trabecular meshwork stem cells

Background:Loss of cells in the human trabecular meshwork(TM)has been reported with ageing and in glaucoma.This study aims to identify,quantify and determine the age-related changes of human TM stem cells(TMSCs).Methods:Isolation of TM cells/paraffin sectioning was carried out using human corneoscleral rings and whole globes.The TM cells/sections were immunostained for the stem cell markers ATP-binding cassette protein G2(ABCG2),nerve growth factor receptor p75 and AnkyrinG(AnkG).Images were acquired using Leica SP8 confocal microscope.The isolated cells were analyzed for two parameters-ABCG2 expression and nucleus to cytoplasmic ratio(N/C ratio).The total number of TM cells and those positive for ABCG2 and p75 in each section were quantified.Spearman rank order correlation was used to determine the association between age and the cell counts.Results:The TMSCs were identified based on two parameters-high ABCG2 expression and high N/C ratio>0.7.These stem cells were also positive for p75 and AnkG.The TMSC content based on the two parameters was 21.0±1.4%in<30 years age group,12.6±6.6%in 30–60 years and 4.0±3.5%in>60 years.The stem cells with high ABCG2 and p75 expression were restricted to the Schwalbe’s line region of the TM.A significant correlation was observed between the reduction in TMSC content and TM cell count during ageing.Conclusion:The human TMSCs were identified and quantified based on two parameter analysis.This study established a significant association between age-related reduction in TMSC content and TM cell loss.

线粒体与青光眼关系的研究进展

青光眼是以特征性视神经损伤和视野缺损为主要表现的一类眼病,其本质是视网膜神经节细胞(RGC)的损伤。原发性开角型青光眼和原发性闭角型青光眼的原发部位是小梁网,线粒体介导的氧化应激是青光眼患者RGC损伤和人眼小梁网细胞(HTMC)死亡的关键。本文就青光眼患者的线粒体基因改变、HTMC和RGC的线粒体氧化应激机制、中药通过氧化应激途径治疗青光眼的机制以及线粒体自噬与青光眼的关系展开综述,从线粒体介导的分子机制层面去认识青光眼的病理机制,为研发青光眼新的药物靶点提供方向。

Experimental study of growth of trabecular cells on the filters and hydraulic conductivity

Objectives To search the method of culturing human trabecular cells (HTC) on a filter support so as to provide a model to study the hydraulic conductivity of HTC in vivo.Methods The third passage of HTC was cultured on a nylon filter; after that we measured the rate of different irrigations through the filter with HTC [Lp, μl/(min· mm Hg·cm 2)]. Results HTC could continuously grow on the filters. The normal Lp was 10.45 μl/(min·mm Hg·cm 2). Irrigated by the solution of epinephrine (EPI) or dexamethasone (DEX), Lp of HTC were higher than that in controls of the same cultural time, while after being exposed to DEX for a few days, Lp was significantly decreased.Conclusions (1) More information of hydraulic conductivity and effects of pharmacologic agents on HTC could be got from the dynamic filtery model; (2) EPI could improve the conductivity of HTC while DEX could have the same effect in early period.

血管紧张素Ⅱ对体外培养的牛眼小梁细胞微管及微丝动力学影响的研究

目的 研究血管紧张素 (angiotensin ,Ang )对体外培养的牛眼小梁细胞骨架的动力学影响 ,探讨 Ang 对房水的调节作用及在青光眼发病机制中的意义。方法 (1)牛眼小梁细胞的体外培养 ,应用免疫组化方法 (NSE, 因子相关抗原染色 )细胞鉴定 ,光学及电子透射显微镜对细胞进行形态学及生长特性的观察 ;(2 )以含不同浓度 Ang -的培养液孵育小梁细胞 12～ 2 4h,(终浓度分别是 10 - 5、10 - 6、10 - 7m ol· L- 1 )。免疫组化法 (SABC)对α- sm ooth- actin、tubulin-α染色。结果以计算机图像分析系统分析 ,并统计学检验。结果 牛眼小梁细胞培养成功 ,成活率 80 %～ 10 0 % ,上皮型为主 ,Ang 产生明显的细胞收缩效应 ,actin是收缩的主要效应器。结论 体外培养牛眼小梁细胞技术是研究小梁细胞特性的重要实验技术。 Ang 可以引起小梁细胞的浓度依赖性的收缩 ,提示 Ang可能是房水排出途径的调节因子之一 。

转化生长因子-β_2对牛眼小梁细胞吞噬功能的影响

观察转化生长因子 β2 (transforming growth factor- β2 ,TGF- β2 )对体外培养牛眼小梁细胞吞噬功能的影响。体外培养牛眼小梁细胞经 0、 0 .32、 1.0 0、 3.2 0 ng/ m l TGF- β2 处理 2 4h后 ,加入乳胶微粒 ,2 4h后作瑞氏染色 ,光镜下数取相邻 2 0个细胞中的微粒数。结果发现 :0 .32、 1.0 0、 3.2 0 ng/ ml TGF- β2 可促进牛眼小梁细胞吞噬乳胶微粒 ,与对照组比较有显著差异 ;同时呈现明显的量效关系。提示 TGF- β2

人眼小梁细胞的体外培养与细胞骨架和细胞连接的研究

目的建立体外培养的人小梁(human trabecular meshwork,HTM)细胞,并观察小梁细胞细胞骨架与细胞连接的形态学特征。方法体外培养的HTM细胞传代后,免疫组化方法鉴定,电镜观察小梁细胞的超微结构,激光共聚焦显微镜观察actin、vinculin、β-catenin的表达。结果通过光镜、透射电镜、扫描电镜和免疫组化的方法证实体外培养的细胞为人眼小梁细胞。人眼小梁细胞的细胞骨架与细胞连接丰富。结论体外培养的小梁细胞细胞骨架结构明显,证明该细胞有收缩能力;β-连接素与纽蛋白着染明显,说明体外培养的小梁细胞,其细胞与细胞间、细胞与细胞基质间的连接仍具有活体小梁细胞的特性。

过氧化氢对人小梁细胞GRP78和CHOP表达的影响

目的:观察H_2O_2对人小梁细胞(HTCs)内质网应激相关蛋白GRP78、CHOP及其mRNA表达的影响。方法:实验分为对照组、H_2O_26 h组、H_2O_212 h组和H_2O_224 h组,分别以600μmol/L H_2O_2作用于HTCs 0、6、12及24 h后,采用Real-time PCR和Western blot检测细胞中CHOP、GRP78 mRNA和蛋白的表达。结果:对照组无GRP78和CHOP的表达;H_2O_2处理后HTCs GRP78、CHOP mRNA及蛋白的表达水平均升高,且随着H_2O_2作用时间的延长,GRP78、CHOP mRNA及蛋白的表达水平升高(P均<0.05)。结论:H_2O_2可诱导HTCs发生内质网应激,从而参与HTCs的氧化损伤。

青光安颗粒剂对自发性青光眼模型小鼠的降眼压作用及房水动力学的影响

背景研究表明青光眼的眼压增高与转化生长因子-β（TGF-β）促进小梁网细胞外基质的堆积以及黏附分子CD44导致房水排出阻力增加有关。传统中药青光安颗粒剂是中医临床上常用的降跟压药物，其是否通过小梁网通路发挥作用尚不清楚。目的研究青光安颗粒剂对自发性青光眼模型DBA／2J小鼠房水动力学的影响及其作用机制。方法选取lO只眼压正常的3月龄雌性DBA／2J小鼠作为对照组，采用计算机随机数字分配法将20只9月龄自发性眼压升高的DBA／2J小鼠随机分为高眼压组和青光安组，青光安组小鼠采用2．5g／kg复方青光安颗粒剂灌胃，每天2次，连续15d，对照组和高眼压组小鼠以相同剂量生理盐水灌胃。采用经前房注入／抽吸系统进行眼压测量，分别以2．5、5．0μl／min的速率继续灌注，计算房水流畅系数（c）值和房水流出阻力（R）值。将3月龄DBA／2J小鼠60只按计算机随机数字分配法分为高剂量青光安组、中剂量青光安组、低剂量青光安组和空白对照组，每组各15只，分别用25．00、12．50和6．25g／kg青光安颗粒剂灌胃，空白对照组用等容量生理盐水灌胃，给药后7d麻醉条件下提取各组小鼠含青光安药物血清和空白对照动物血清，然后收集各组小鼠含小梁网巩膜组织进行培养，采用纤连蛋白（FN）、层黏连蛋白（LN）和神经元特异性烯醇化酶（NSE）免疫组织化学染色鉴定小梁网细胞。终质量浓度为0、5、10、20、50和100ng／ml的TGF-β处理小梁网细胞24h，采用MTT比色法检测各组细胞活性；用不同质量浓度含药血清培养经20ng／mlTGF-β处理的小梁细胞，分别于培养后24、48和72h采用ELISA法检测细胞上清液中TGF-β2受体质量浓度，采用Westernblot法检测各组小梁网细胞中CD44蛋白的相对表达量。结果对照组、高眼压组和青光安组小鼠在2．5td／min和5．0μl／min灌注速率下眼压明显不同，高眼压组小鼠眼压值明显高于青光安组和对照组，青光安组小鼠c值较高眼压组明显降低（1．08±O．36％2．35±1．34），R值明显增加（1．05±O．47 vs 0．64±0．55），差异均有统计学意义（均P〈O．01）。原代培养的细胞呈长梭形，FN、LN和NSE均呈阳性表达。5、10和20ng／mlTGF．p处理组细胞活力值明显低于0ng／mlTGF-β处理组细胞，差异均有统计学意义（均P〈0．05）。与空白对照组比较，TGF．母组小梁网细胞上清液中TGF-β2受体质量浓度和细胞中CD44蛋白相对表达量均明显升高，差异均有统计学意义（均P〈O．01）；细胞培养后24、48和72hTGF-β高剂量含药血清组细胞上清液中TGF-β2受体质量浓度和细胞中CD44相对表达量均明显低于TGF-β组和TGF-B＋低剂量含药血清组，差异均有统计学意义（均P〈0．01）。结论青光安颗粒剂通过影响房水动力学因素达到降低青光眼眼压的作用。青光安含药血清能明显降低体外培养的鼠小梁网细胞中TGF-β2受体和CD44的表达。

人眼小梁网细胞的体外培养

目的 探讨体外培养人眼小梁网细胞的方法并观测其生物学特性。 方法 采用组织块培养方法进行体外细胞培养 ,二甲基噻唑二苯基四唑溴盐比色法、倒置显微镜、透射电镜 ,免疫细胞化学等方法观察体外培养细胞的生物学特性并进行鉴定。 结果 组织块培养 7～ 15 d后可见细胞从其边缘向外生长。倒置显微镜下成梭形、圆形、椭圆形、并有多个突起 ;电镜可见小梁网细胞表面有较多微绒毛 ,细胞间缝隙连接 ,胞质内见很多次级溶酶体。免疫细胞化学检测纤维黏连蛋白、层黏连蛋白和神经元特异性烯醇化酶染色阳性。这些特征可与角膜内皮细胞和巩膜成纤维细胞相鉴别。 结论 组织培养方法能够成功体外培养人眼小梁网细胞。

体外培养牛眼小梁细胞表达转化生长因子β及其受体蛋白

目的 从蛋白质水平了解体外培养牛眼小梁细胞是否表达转化生长因子 β(TGF β)及其受体 (TGF βR)。 方法 采用Supervision法对体外培养第 3代牛眼小梁细胞进行TGF β1,TGF βR免疫组化检测。 结果 小梁细胞TGF β ,-β2 和TGF βRⅠ ,RⅡ ,RⅢ免疫组化染色的阳性信号分别位于胞浆内和胞膜上 ,阴性对照未见阳性信号。 结论 牛眼小梁细胞表达TGF β ,TGF βR蛋白。较之猪眼小梁细胞 ,牛眼小梁细胞更适用于今后研究TGF β及其受体与原发开角型青光眼发病学的关系。

低渗诱导人小梁细胞容积敏感性氯电流的电生理学特点

目的:探讨与小梁细胞容积调节有关的氯通道电流的生理学特点,阐明该电流与容积敏感性氯通道电流的关系。方法:体外培养人小梁细胞,全细胞膜片钳记录等渗和低渗条件下小梁细胞膜上的氯通道电流,观察该电流特点。低渗状态下在浴液中加入氯离子通道阻断剂NPPB和他莫昔芬及ATP后记录电流的变化。结果:在等渗条件下,仅观察到微弱且稳定的背景电流,在+100和-100mV电压钳钳制下,外向和内向电流密度值(pA/pF)分别为1.48±0.36和-0.91±0.10(n=6)。低渗刺激后,细胞迅速肿胀,电流较等渗时明显增大,呈外向优势,外向和内向电流密度值(pA/pF)分别为19.94±0.87和-6.53±0.41(n=6,P<0.01)。在低渗状态下的浴液中分别加入NPPB(100mol.L-1)、他莫昔芬(50mol.L-1)和ATP(2μmol.L-1)20min后,外向和内向电流密度值(pA/pF)分别为7.61±0.50和-3.70±0.25、8.21±0.63和-3.83±0.25、9.08±0.67和3.89±0.22,均较低渗时明显减小(P<0.01)。结论:细胞外低渗诱导人小梁细胞产生呈外向优势氯电流,此电流对细胞容积的改变敏感,可被NPPB、他莫昔芬和ATP抑制,该电流符合容积敏感性氯电流的特征。

转化生长因子β_2诱导人小梁细胞凋亡的体外研究

目的 观察转化生长因子 β2 (transforminggrowthfactor β2 ,TGF β2 )是否能诱导体外培养人眼小梁细胞 (humantrabecularmeshworkcells,HTMC)发生凋亡。方法 将体外培养的第 3～ 5代人眼小梁细胞分为对照组和实验组 ,实验各组分别经 0 .32、1.0 0、3.2μg·L-1TGF β2 作用 4 8h后 ,分别用透射电镜、TUNEL法和流式细胞术检测细胞凋亡的诱导情况。结果 透射电镜观察发现凋亡细胞典型的形态学变化———核染色质凝集边集 ,凋亡小体形成等。TUNEL法检出发生DNA片段化的HTMC。经流式细胞术定量分析 ,不同浓度TGF β2 处理的HTMC凋亡细胞百分数分别为 ( 2 .79± 0 .4 4 ) %、( 4 .4 3±1.17) %、( 9.6 0± 2 .0 5 ) % ,其中 1、3.2 μg·L-1TGF β2 处理组与对照组 ( 1.4 1± 0 .34) %相比分别有显著差异和极显著差异。结论 TGF β2 能诱导体外培养HTMC发生凋亡 ,推测其可能参与了原发性开角型青光眼患者和正常人衰老过程中HTMC数目的减少。