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

Primary open- angle glaucoma (POAG) is aleading cause of blindness,which involves optic neu-ropathy accompanied by characteristic visual field de-fects and is often associated with elevated intraocularpressure due to disturbance ofaqueoushumor outflowthrough the trabecularmeshwork (TM) .The patho-physiology of the TM in POAG has been character-ized by an increasein extracellularmatrix componentsand a decrease in the number of TM cells[1,2 ] .It hasbeen found that,compared with the non- P…     

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.     

体外培养人眼小梁细胞黏附分子CD44s的表达

目的 探讨黏附分子CD44s在体外培养人眼小梁细胞（HTC）的表达及其与小梁细胞结构和功能的关系。方法 对体外培养的HTC应用流式细胞术和RT-PCR法检测CD44s的表达。结果 HTC高表达CD44s黏附分子，阳性细胞数达92％以上，而且均表达CD44s mRNA。结论 体外培养的HTC表达CD44s黏附分子。CD44s与其配体透明质酸（HA）的协同性调节HTC的内环境并在维持其结构和功能方面起重要作用。     

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。     

IGF-1 mRNA expression of adult rat thyroid cell cultured in vitro

Withageincreasing ,fibrosis,cellinfiltration ,folliclevariation ,nodformation ,thyroidweightloss ,lessthyroidhormoneoccursinthyroid .Inrecentyears ,thereweremanyresearchesfortherelationshipbetweenthyroidfunctionandagingaswellasthethy roidfunctioninpostponingaging[1] ,butthestudyofmolecularlevelonthechangeofthyroidcellandageingisless .IGF 1distributesinallorgansandtissues .IGF 1ofthyroidcellplaysanimportantroleinthyroidfunctionregulation[2 ] .Ourexperimentinvestigatesthelawofage relatedchange…     

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…     

电压门控性氯通道3在高眼压模型大鼠小梁网中的表达

目的: 检测电压门控性氯离子通道3(ClC-3)在大鼠小梁网中的表达情况,探讨其在青光眼发病机制中的可能作用。方法: 30只健康雄性大鼠。左眼作为正常对照,右眼前房内注射玻璃酸钠,每周1次,连续10周,制备高眼压模型,每次全麻后玻璃酸钠注射前采用Tono-Pen XL眼压计测量双眼眼压并记录。实验分为正常对照组、玻璃酸钠注射3周组和10周组。分别取3组大鼠的小梁网组织,采用RT-PCR法检测ClC-3mRNA表达水平,免疫组织化学法检测ClC-3蛋白的表达。结果: 玻璃酸钠注射后1、3和10周,大鼠右眼眼压均较注射前增高(P<0.01),注射后1周眼压值较注射前轻微增高,注射后3周眼压较注射前明显增高,但眼压值未达到高眼压成模标准,注射后10周眼压值较注射前明显增高,达到高眼压成模标准。RT-PCR法检测,与正常对照组比较,玻璃酸钠注射3周组大鼠小梁网组织中ClC-3 mRNA表达水平明显增高(t=7.88,P<0.05);玻璃酸钠注射10周组大鼠小梁网组织中ClC-3 mRNA表达水平明显降低(t=15.93,P<0.05)。免疫组织化学检测,3组大鼠小梁网组织中ClC-3蛋白表达均为阳性,玻璃酸钠注射3周组大鼠小梁网组织中ClC-3蛋白表达强度较正常对照组大鼠增强,玻璃酸钠注射10周组大鼠小梁网组织中ClC-3蛋白表达强度较正常对照组大鼠减弱。结论: 大鼠小梁组织中存在ClC-3的表达,ClC-3可能与小梁网的病理调节有关联,进而参与青光眼的发病。     

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 (HT-MCs) in vitro, and to evaluate the inductive role of heroin 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.     

Effect of CD44 Suppression by Antisense Oligonucleotide on Attachment of Human Trabecular Meshwork Cells to HA

The effects of suppression of CD44 by CD44-specific antisense oligonucleotide on attachment of human trabecular meshwork cells to hyaluronic acid (HA) were observed and the possible relationship between CD44 and primary open-angle glaucoma (POAG) investigated. CD44-specific antisense oligonucleotide was delivered with cationic lipid to cultured human trabecular meshwork cells. The expression of CD44 suppressed by CD44-specific antisense oligonucleotide was detected by RT-PCR and Western blotting. The effect of CD44 suppression by specific antisense oligonucleotide on attachment of trabecular meshwork cells to HA was measured by MTT assay. Results showed that expression of CD44 was suppressed by CD4 , specific antisense oligonucleotide. Antisense oligonucleotide also suppressed the adhesion of human trabecular meshwork cells to HA in a concentration dependent manner. It was concluded that attachment of human trabecular meshwork cells to HA was decreased when CD44 was suppressed by specific antisense oligonucleotide. CD44 might play a role in pathogenesis of POAG by affecting the adhesion of trabecular meshwork cells to HA.     

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).     

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.     

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.     

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…     

Existence of heme oxygenase-carbon monoxide-cyclic guanosine monophosphate pathway in human trabecular meshwork cells in vitro

Summary 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 his pathway, HTMCs of the third to fourth generation were culturedin vitro. Reverse transcipase-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. LI Tao, male, born in 1976, Doctor in Charge     

初生及成年牛眼小梁网,睫状体及视网膜中原癌基因的表达

目的：研究正常成年及新生牛眼组织原癌基因的表达水平。方法：分离牛眼组织，提取ＲＮＡ，应用缝隙印迹杂交和Ｎｏｒｔｈｅｒｎ印迹杂交技术，分析眼组织中Ｎ－ｒａｓ，Ｃ－ｍｙｃ和Ｃ－ｆｏｓ基因的表达水平。结果：Ｎ－ｒａｓ基因在牛眼小梁网、睫状体及视网膜神经层均有表达，初生及成年牛间无差异，Ｃ－ｍｙｃ基因在这三种组织中有较高表达，且初生牛比成年牛眼表达水平高。Ｃ－ｆｏｓ基因不表达。结论：正常眼组织中原癌基因的     

垂体腺瘤中干细胞样细胞的培养及初步鉴定

目的 检测垂体腺瘤组织中干细胞标志物的表达情况,培养并初步鉴定垂体瘤干细胞。 方法 提取42例液氮冻存的垂体腺瘤组织标本中的总RNA,通过RT-PCR方法检测瘤组织中Nestin的表达情况;通过免疫荧光法检测无血清条件下培养的垂体瘤细胞球中干细胞标志物(Nestin、Sox2)的表达情况。 结果 42例肿瘤组织标本中,Nestin mRMA阳性表达37例;无血清培养条件富集培养的悬浮细胞球中均可通过免疫荧光法检测出Nestin、Sox2的表达。 结论 垂体腺瘤组织中存在干细胞标志物的表达,通过无血清培养亦可培养出垂体瘤干细胞。