Regulatory Effect of Dexamethasone on Aquaporin-1 Expression in Cultured Bovine Trabecular Meshwork Cells

Itiswell knownthattheglucocorticoidcancauseintraocularhypertensionafterlong termlocalorsystematicapplication.Thechangesinopticpa pillaanddefectofvisualfieldwillresultifthehighintraocularpressurepersistsforsometime.Nowa days,withthewidespreaduseofglucocorticoidinclinicalpractice,thecasesofsecondaryglaucomainducedbyglucocorticoid(GIG)areincreasingdramatically.Althoughmanyinvestigationscon cerningGIGhavebeenconducted,theexactmech anismremainsunclear.Recentintroductionofaquaporinsconceptionandt…     

Antagonistic effects of tranilast on proliferation and collagen synthesis induced by TGF-beta2 in cultured human trabecular meshwork cells.

Whether tranilast had antagonistic effect on proliferation inhibition and collagen synthesis promotion induced by TGF-beta2 in cultured human trabecular meshwork cells was investigated. Suspension of 1 x 10(4) 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 microg/ml (control), 12.5 microg/ml, 25 microg/ml, 50 microg/ml tranilast with 3.2 ng/ml TGF-beta2 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 microg/ml (q'= 3.23, P< 0.05), 50 microg/ml (q'=4.70, P<0.01) tranilast significantly antagonized the decrease of the A values induced by TGF-beta2 in the cultured human trabecular meshwork cells. In comparison with the control group [817.37+/-124.21 cpm/10(4) cells], 12.5 microg/ml (620.33+/-80.46 cpm/10(4) cells, q'= 4.26, P<0.05), 25 microg/ml (594.58+/-88.13 cpm/10(4) cells, q'=4.81, P<0.01), 50 microg/ml (418.64+/-67.90 cpm/10(4) 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-beta2 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-alpha2 in the cultured human trabecular meshwork cells.     

Apoptosis of human trabecular meshwork cells induced by transforming growth factor-β<Subscript>2</Subscript><Emphasis Type="Italic">in vitro</Emphasis><Emphasis Type="Italic">in vitro</Emphasis>

Summary Whether transforming growth factor-β₂ (TGF-β₂) induces apoptosis of human trabecular meshwork cells was investigatedin vitro. Cultured 3–5 passage human trabecular meshwork cells were treated with 0 (control). 0. 32. 1, 3. 2 ng/ml TGF-β₂ 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 character istic morphologic changes of apoptotic cells were observed under transmission electron microscopy. DNA fragmentation of human trabecular meshowork 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-β₂ with the difference being significant between experimental group and control group [(1.41±0.34)%]. It was concluded that TGF-β₂ can induce apoptosis of human trabecular meshwork, cellsin 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. CAO Yang, male, born in 1972, M. D., Ph. D., Associate Professor This project was supported by a grant from the National Natural Sciences Foundation of China (No. 38970758).     

Antagonistic Effects of Tranilast on Proliferation and Collagen Synthesis Induced by TGF-β2 in Cultured Human Trabecular Meshwork Cells

Whether tranilast had antagonistic effect on proliferation inhibition and collagen synthesis promotion induced by TGF-β2 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-β2 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 3 H-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-β2 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 3 H-proline into the cultured human trabecular meshwork cells promoted by TGF-β2 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-β2 in the cultured human trabecular meshwork cells.     

A Study of Toxicity of 5-Fluorouracil on Bovine Trabecular Meshwork Cells in Vitro

5- fluorouracil( 5 - Fu) is widely used as an ad-junct in the surgery of ocular diseases such as glauco-ma[1-3 ] ,proliferative vitreoretinopathy[4 ] ,etc.Be-cause of its cytotoxicity,itmay cause corneal epithe-lial defects,conjunctival wound leak[5,6] in filteringsurgery or othercomplications.The trabecularmesh-work cells in the patients with glaucoma may alreadyhave sustained some previous damage,so it is crucialto make sure whether 5 - Fu would cause further in-jury of the cells.Therefore,t…     

Effect of transforming growth factor-β2 on phagocytosis in cultured bovine trabecular meshwork cells

Summary The effect of transforming growth factor-β₂ (TGF-β₂) on phagocytosis in bovine trabecular meshwork cellsin 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-β₂ 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-β₂ 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-β₂ significantly increased the number of the latex beads phagocytosed by cultured bovine trabecular meshwork cells in a dose-dependent manner. TGF-β₂ could promote the phagocytosis of bovine trabecular meshwork cellsin 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. This project was supported by a grant from the National Natural Science Foundation of China (No. 38970758).     

Effect of Dexamethasone and Aquaporin-1 Antisense Oligonucleotides on the Aquaporin-1 Expression in Cultured Human Trabecular Meshwork Cells

The changes in the expression of aquaporin-1 （AQP1） mRNA and protein in cultured human trabecular meshwork （HTM） cells treated with dexamethasone and transfected with antisense oligonucleotides （AS-ODN） were studied, and the implication of AQP1 regulation in corticosteroid-glaucoma and the possibility of AS-ODN inhibiting the AQP1 expression were evaluated. The cultured HTM cells in vitro were treated with different concentrations of dexamethasone and transfected with oligonucleotides for 5 days respectively. Then, total RNA and protein of HTM cells were extracted. The changes of AQP1 mRNA and protein were demonstrated qualitatively and quantitatively by RT-PCR and Western blot. Band intensities were detected by imaging analysis. There was a parallel relationship between the results of RT-PCR and those of Western blot. The expression levels of AQP1 mRNA and protein in dexamethasone-treated groups were increased initially and decreased later as dexamethasone concentration was stepped up. In the 0.04 μg/mL and 0.4 μg/mL groups, the levels of AQP1 were higher than in control group （0 μg/mL）. In the 4 μg/ mL and 40 μg/mL groups, the AQP1 expression levels were lower than in control group. AS-ODN could down-regulate the expression of AQP1 mRNA and protein in a dose-dependent manner. At 5 μg/mL, down-regulation efficiency reached the maximum. There was no statistically significant difference in the expression of AQP1 mRNA and protein between all sense oligonucleotides groups and control group. It was suggested that dexamethasone may induce the changes of the AQP1 expression in HTM cells to be involved in the occurrence of corticosteroid-glaucoma. AS-ODN can down-regulate the AQP1 expression in HTM cells to some extent.     

地塞米松对低渗诱导的人小梁细胞容积敏感性氯电流的影响

目的：分析地塞米松对低渗诱导的人小梁细胞容积敏感性氯电流的影响,探讨容积敏感性氯通道(VACC)在激素性青光眼(GIG)发病中的可能机制。方法：人小梁细胞悬液接种于直径35 mm塑料培养皿中单层生长,按培养液中是否含地塞米松分组,即正常培养液组和地塞米松培养1、3和7 d组,共4组,采用全细胞膜片钳技术分别记录4组细胞在低渗状态下的氯电流密度值,比较各组数值的差异。结果：正常培养液组小梁细胞低渗刺激后,在+100和－100 mV电压钳钳制下,外向和内向电流密度值分别为（19.94±0.87)和(－6.53±0.41)pA/pF。地塞米松培养1、3和7 d组的小梁细胞低渗刺激后,在+100和－100 mV电压钳钳制下,外向和内向电流密度值分别为（19.39±1.40)和(－6.42±0.28)pA/pF、（17.97±2.35)和(－5.82±0.94)pA/pF、（17.16±1.16)和(－5.65±0.43)pA/pF。地塞米松培养1 d组小梁细胞较正常培养液组低渗刺激后的外向和内向电流密度值减小,但差异无统计学意义（P＞0.05）;地塞米松培养3和 7 d组小梁细胞较正常培养液组小梁细胞低渗刺激后的外向和内向电流密度值明显减小（P＜0.05）。结论：地塞米松导致体外培养的正常人小梁细胞容积敏感性氯电流密度值降低,进而影响小梁细胞容积调节,可能是导致GIG房水流出阻力增加的原因之一。     

Growth inhibition and apoptosis induction in human hepatoma cells by tanshinone II<Subscript>A</Subscript>

Summary In order to study the effect of tanshinone II_A on growth and apoptosis in human hepatoma cell line BEL-7402in vitro, the human hepatoma cell line BEL-7402 was treated with tanshione I_A at various concentrations for 72 h. Growth suppression was evaluated by MTT assay; apoptosis-related alterations in morphology and biochemistry were ascertained under cytochemical staining (Hoechst 33258), transmission electron microscopy (TEM), and DNA agarose gel electrophoresis. Apoptotic rate was quantified by flow cytometry (FCM). The results showed that Tanshinone II_A could inhibit the growth of hepatoma cells in a dose-dependent manner, with IC₅₀ value being 6.28 μg/ml. After treatment with 1–10 μg/ml tanshione II_A for 72 h, BEL-7402 cells apoptosis with nuclear chromatin condensation and fragmentation as well as cell shrinkage and the formation of apoptotic bodies were observed. DNA ladder could be demonstrated on DNA electrophoresis. FCM analysis showed hypodiploid peaks on histogram, and the apoptotic rates at 5 μg/ml concentration for 12 h, 24 h, 36 h, 48 h and 72 h were (2.32±0.16)%, (3.01±0.35)%, (3.87±0.43)%, (6.73±0.58)% and (20.85±1.74)% respectively, which were all significantly higher than those in the control group (1.07±0.13)%. It is concluded that Tanshione II_A could induce human hepatoma cell line BEL-7402 apoptosis, which may be related to the mechanism of growth inhibition. TANG Zhongzhi, male, born in 1966, Doctor in Charge This project was supported by a grant from Natural Sciences Foundation of Hubei Province (No. 2000J064).     

人眼小梁组织及体外培养的人眼小梁细胞SPARC mRNA及其蛋白表达

目的:研究人眼小梁组织(TM)及体外培养的人眼小梁细胞(TMCs)是否可以表达酸性富含胱氨酸分泌型蛋白(SPARC) mRNA及SPARC蛋白. 探讨SPARC蛋白在原发性开角型青光眼(POAG)发病机制中的作用. 方法:分别采用RT-PCR和Western-Blot方法,对小梁组织和细胞进行检测,用抗SPARC蛋白免疫荧光染色方法对细胞进行染色. 结果:组织和细胞RT-PCR均在300 bp处出现预期的电泳条带,组织和细胞Western-Blot在Mr为43×103处出现蛋白条带. TMCs抗SPARC免疫荧光染色表现为胞质均匀着色. 结论:TM组织及体外培养的人眼TMCs均可表达SPARC mRNA及其相关蛋白,并起着调节细胞外基质(ECM)的生成和降解作用.     

人眼组织小梁网细胞的体外培养及鉴定的新方法

目的探讨人原代小梁网细胞的体外培养,以及利用其特性建立一种鉴定小梁网细胞的新方法。方法从眼球破裂伤病人的眼球以及角膜移植后剩余的角膜环分离出小梁网组织,利用组织块贴壁法以及消化法对人原代小梁网细胞进行体外培养。倒置显微镜下观察细胞生长状态并利用CCK8法检测其生长速率。利用细胞免疫荧光技术对所培养的细胞进行纤维连接蛋白、Ⅳ型胶原蛋白、层黏连蛋白、水通道蛋白-1等蛋白的染色鉴定。并利用100 nmol/L地塞米松对所培养的细胞诱导10 d,通过荧光定量PCR和western blotting方法检测myocilin的表达水平以确定所培养的细胞是否为小梁网细胞。结果小梁网组织块贴壁培养1~2周后,开始有细胞从组织块旁向外长出,并逐渐增多。传代后小梁网细胞在第1~4天生长较快,第5~7天生长速度有所减慢,但依然显著高于第4天（P < 0.01）。所培养的细胞纤维连接蛋白、Ⅳ型胶原蛋白、层黏连蛋白、水通道蛋白-1的免疫荧光染色均呈阳性。地塞米松诱导后,与对照组相比,小梁网细胞中myocilin mRNA和蛋白表达水平均明显上升（P < 0.01）。结论本实验中所培养的细胞通过对其特点进行检测,确定所培养的细胞为人原代小梁网细胞。     

Apoptosis of human trabecular meshwork cells induced by transforming growth factor-beta2 in vitro.

Whether transforming growth factor-beta2 (TGF-beta2) 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-beta2 for 48 h and divided into control group and experimental group. The apoptosis of human trabecular meshwork cells was examined by transmission 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-beta2 with the difference being significant between experimental group and control group [(1.41 +/- 0.34)%]. It was concluded that TGF-beta2 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.     

氧化应激对猪眼小梁细胞内皮细胞白细胞黏附分子-1表达的影响

目的 研究氧化应激对猪眼小梁细胞内皮细胞白细胞黏附分子-1（ELAM-1）表达的影响,探讨氧化应激诱导的ELAM-1的表达与白细胞介素1α（IL-1α）的关系。方法原代培养的猪眼小梁细胞经过血清饥饿培养后,用不同浓度白细胞介素-1受体拈抗剂（IL-1rα）处理或直接用1mmol／L的H2O2刺激后,用免疫细胞化学法检测小梁细胞ELAM-1的表达。结果 H2O2处理的原代培养的猪眼小梁细胞中ELAM-1表达呈阳性,高浓度（180μg／ml和600μg／m1）拈抗剂IL-1rα预处理的猪眼小梁细胞ELAM-1表达呈阴性。结论 氧化应激可诱导猪眼小梁细胞表达ELAM-1。在体外细胞培养体系,高浓度的IL-1α可拮抗氧化应激对ELAM-1表达的作用。     

SRB法和MTT法检测PDGF-BB对牛眼小梁细胞增殖的影响

目的探讨血小板源性生长因子-BB(platelet-derived growth factor-BB,PDGF-BB)对体外培养牛眼小梁细胞增殖的影响及意义。方法分别用不同浓度的PDGF-BB(0 ng/ml、5.0 ng/ml、12.5 ng/ml、25.0 ng/ml)刺激第三代的牛眼小梁细胞。24小时后收集细胞,分别采用SRB法和MTT法检测PDGF-BB对牛眼小梁细胞增殖的影响。结果 SRB法和MTT法均显示,随着PDGF-BB浓度的增加,牛眼小梁细胞增殖能力加强。结论在体外培养的条件下,PDGF-BB可以促进牛眼小梁细胞增殖,可能对降低眼内压具有重要作用。     

Measurement of platelet-activating factor in human blood by high performance thin layer chromatography and its clinical application

Summary Platelet-activating factor (PAF) present in the blood of the patients with chronic pulmonary heart disease and asthma has been detected by high performance thin layer chromatography (HPTLC). The patients with chronic pulmonary heart disease accompanied by carbon dioxide retention (PaCO₂>6.67 kPa) have a higher level of PAF in blood (0. 75 ±0. 27 μg/ml) than those who have no carbon dioxide retention (PaCO₂<6.67 kPa, PAF 0.41 ±0. 25 μg/ml) and those in the normal control group (0.45 ±0.20 μg/ml), withP<0.05 in all. The patients with asthma have a higher PAF in blood (0.83±0. 05 μg/ml) than those in the control group (P < 0.005). These findings suggest that PAF plays an important role in episodes of chronic pulmonary heart disease and asthma.     

Effects of hydrostatic pressure on cultured bovine trabecular meshwork cells

Elevationofintraocularpressureisoneofthemaincharacteristicsofglaucoma,whichcausesdamagetovariousoculartis-sues[1'2].Theinvestigationofpressureontrabecularmeshworkcellswasseldomre-ported,andwaslimitedtoinvivoresearch-es[3'4],whichinvolvedmanyinfluencingfactors.Therefor,weculturedbovinetra-becularmeshwork(BTM)cellsinvljroandsubjectedthecellstodifferentlevelsofhy-drostaticpressuretoexploreitspossibleef-fects.1METHODS1.1ProcedureofBTMCellCuItureTheprocedureofisolationofbovinetrabeculumandth…     

Effect of Interleukin-1β on IA and IK Currents in Cultured Murine Trigeminal Ganglion Neurons

To investigate the effect of interleukin-1β (IL-1β) on IA and IK currents in cultured murine trigeminal ganglion (TG) neurons, whole-cell patch clamp technique was used to record the IA and IK currents before and after 20 ng/mL IL-1β perfusion. Our results showed that 20 ng/mL IL-1β inhibited IA currents (18.3±10.7)% (n=6, P<0.05). IL-1β at 20 ng/mL had no effect on G-V curve of IA but moved the H-infinity curve V0.5 from -36.6±6.1 mV to -42.4±5.2 mV (n=5, P<0.01). However, 20 ng/mL IL-1β had effect on neither the amplitude nor the G-V curve of IK. IL-1β was found to selec- tively inhibit IA current in TG neurons and the effect may contribute to hyperalgesia under various in- flammatory conditions.     

重组人骨形态发生蛋白7对原发性开角型青光眼小梁网细胞增殖的影响

目的 建立原发性开角型青光眼(POAG)小梁网细胞体外原代培养体系; 分析不同终浓度重组人骨形态发生蛋白7(rhBMP7)对POAG小梁网细胞增殖的影响; 探讨rhBMP7与POAG发生、进展的相关性。 方法 取术中切除的带小梁网组织块(未使用丝裂霉素C),进行体外原代及传代培养,取3代小梁网细胞,在CFDA SE标记后,分别加入终浓度为0(对照组),20,50,80,100,200 ng/mL的rhBMP7无血清培养基,采用CCK8、荧光显微镜、流式细胞仪等方法检测POAG小梁网细胞增殖情况。 结果 细胞经传代培养,经鉴定为POAG小梁网细胞; 采用CCK8法检测发现:经终浓度为0(对照组),20,50,80,100,200 ng/mL的rhBMP7处理后,POAG小梁网细胞吸光度(OD值)分别为:(0.561 2±0.026 9),(0.724 2±0.039 3),(1.416 0±0.016 2),(1.740 4±0.039 2),(1.853 8±0.014 5),(1.936 4±0.054 6); 实验组细胞增殖率分别为1.37%,2.96%,3.70%,3.96%,4.15%; 实验组与对照组及各实验组间增殖率比较,差别具有统计学意义(P<0.05); 荧光显微镜示:随着rhBMP7终浓度的增加,经CFDA SE标记的POAG小梁网细胞荧光染色变浅、细胞量及密度增加; 采用流式细胞仪检测经20,50,80,100,200 ng/mL终浓度rhBMP7干预的POAG患者小梁网细胞,分裂、增殖细胞所占的比例分别为17.85%,18.63%,20.10%,27.45%,72.41%。 结论 运用组织块培养法,可体外原代培养出POAG患者的小梁网细胞; rhBMP7在一定程度上可促进POAG小梁网细胞的增殖,且在一定范围内呈剂量依赖性。     

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.