脂质体介导的CD44反义寡核苷酸对人眼小梁细胞粘附功能的影响

目的：观察脂质体介导的CD44反义寡核苷酸对人眼小梁细胞粘附功能的影响，探讨CD44分子在原发性开角型青光眼(POAG)发病过程中可能的作用。方法：采用硫代修饰的CD44反义寡核苷酸，通过脂质体导人体外培养的人眼小梁细胞，RT—PCR、Western印迹及MTT法观察CD44反义寡核苷酸对人眼小梁细胞CD44基因表达及粘附功能的影响。结果：RT—PCR及Western印迹结果提示：CD44反义寡核苷酸抑制人眼小梁细胞CD44表达。MTT法检测显示：CD44反义寡核苷酸对人眼小梁细胞与细胞外基质透明质酸的粘附起抑制作用且呈浓度依赖性。结论：CD44反义寡核苷酸抑制人眼小梁细胞与细胞外基质透明质酸的粘附，粘附分子CD44可能参与了POAG发病过程。     

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.     

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.     

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.     

压力对体外培养牛眼小梁细胞的影响

目的：观察压力对体外培养牛眼小梁细胞的影响．方法：对体外培养的牛眼小梁细胞施以不同程度的压力，用倒置相差显微镜，光镜，电镜观察细胞形态结构变化，台盼蓝染色观察比较各组细胞活力的差别．结果：当作用于细胞的压力为1．33和2．67kPa(1kPa＝7．5mmHg)，作用时间为48h时，与对照组比较，形态结构无明显变化，台盼蓝计数无显性差异(P＞0．05)，而当压力为5．33kPa作用时间为24h时，细胞形态结构有轻度的损伤，随着压力和作用时间的进一步增加，细胞的损伤程度也进一步加重。结论：小梁细胞承受压力的能力是有限的，压力过高或受压时间过长都会对细胞造成损伤。     

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…     

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…     

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－100 mV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(100 mol/L)、他莫昔芬（50 mol/L）和ATP (2 μmol/L)20　min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。     

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…     

氯通道ClC-2反义寡核苷酸对顺铂作用下神经胶质瘤U251细胞侵袭力的影响

目的：探讨氯通道ClC-2 反义寡核苷酸(AON)抑制ClC-2基因表达对顺铂(DDP)作用下神经胶质瘤U251细胞侵袭力的影响,为ClC-2成为神经胶质瘤综合治疗的新基因靶点提供理论依据。方法：U251细胞按处理方法不同分为对照组（转染无义寡核苷酸）、AON组（转染ClC-2反义寡核苷酸）、DDP组（无义寡核苷酸与顺铂联用）和AON +DDP组（ClC-2反义寡核苷酸与顺铂联用）。应用RT-PCR检测相关基因ClC-2、cyclin D1 、MMP-2、MMP-9 mRNA表达;Transwell 小室侵袭实验检测各组细胞侵袭力;Transwell 小室迁移实验检测各组细胞迁移率。结果：与对照组比较,AON组、DDP组和AON +DDP组ClC-2、cyclin D1 、MMP-2、MMP-9 mRNA表达率降低（P＜0.05）;Transwell 小室侵袭实验中对照组、AON组、DDP组和AON +DDP组侵袭细胞数分别为81.00±4.58、42.01±4.36、48.33±2.52和12.67±5.13;与对照组比较,AON组、DDP组和AON +DDP组穿透细胞数明显降低（P＜0.05）。Transwell 小室迁移实验中AON 组、DDP组、AON +DDP组细胞迁移率（%）分别为72.40±2.20、51.48±1.29、37.87±1.50,与对照组比较均明显降低（P＜0.05）。结论：①转染ClC-2反义寡核苷酸能有效抑制ClC-2 mRNA表达;②抑制ClC-2 mRNA能降低U251细胞的迁移力和侵袭力;③ClC-2 mRNA表达降低可促进顺铂对U251细胞迁移力和侵袭力的抑制作用。     

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。     

Antagonistic effects of trasilast on proliferation and collagen synthesis induced by TGF-\sB<Subscript>2</Subscript> in cultured human trabecular meshwork cellsin cultured human trabecular meshwork cells

Summary Whether tranilast had antagonistic effect on proliferation inhibition and collagen synthesis promotion induced by TGF-\sB₂ 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, 50μg/ml tranilast with 3.2 ng/ml TGF-\sB₂ 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 and³H-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 theA values induced by TGF-\sB₂ in the cultured human trabecular meshowrk 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 (59.4.58±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 of³H-proline into the cultured human trabecular meshwork cells promoted by TGF-\sB₂ 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-\sB₂ in the cultured human trabecular meshwork cells. Da Banghong, female, born in 1973, Resident. This project was supported by a grant from the National Natural Sciences Foundation of China (No. 38970758).     

生存素反义寡核苷酸对人胃癌细胞增殖的抑制作用

目的 探讨生存素（survivin）反义寡核苷酸（ASODN）对人胃癌细胞SGC7901增殖的抑制作用。方法 设计合成特异性靶向生存素的反义寡核苷酸。胃癌细胞株SGC7901分为4组：空白对照组、单纯脂质体对照组、正义链转染对照组、ASODN转染组。作用48h后收获各组细胞。Westernblot法检测各组细胞生存素表达情况，流式细胞仪检测各组细胞凋亡率，MTT法检测各组细胞的生长抑制率。结果 脂质体介导生存素反义寡核苷酸转染后的胃癌细胞出现生存素蛋白表达明显下降；ASODN转染组细胞凋亡率明显高于各对照组（P〈0．05），而各对照组问差异无显著性（P〉0．05）；ASODN转染组细胞的抑制率明显高于各对照组（P〈0．05），而各对照组问差异无显著性（P〉0．05）。结论 生存素反义寡核苷酸转染胃癌细胞能下凋生存素蛋白表达，诱导胃癌细胞凋亡，抑制细胞增殖，具有明显的抗癌作用。     

HES1在小梁网细胞中作用的初步研究

目的 : 利用慢病毒包装系统使人眼小梁网细胞（HTMCs）稳定表达HES1 shRNA并初步探索HES1在HTMCs的作用。 方法: 体外培养HTMCs和HEK293FT细胞并进行传代,同时应用pLKO.1-puro shRNA慢病毒载体构建HES1 shRNA质粒。Lipofectamine 2000慢病毒包装法感染原代HTMCs,使其稳定表达HES1shRNA/Scramble质粒。采用q-PCR 和western blot方法检测HES1shRNA的敲低效果及促纤维化细胞外基质蛋白（ECM）的表达变化;HTMCs增殖和迁移能力分别通过CCK-8细胞活性分析和transwell计数实验进行分析。 结果: 原代细胞经过培养后贴壁生长,呈长梭形,生长状态良好,形态符合HTMCs形态学特征。HES1 shRNA有效下调HES1 的mRNA和蛋白水平表达,P<0.05。同时,HES1 shRNA组促纤维化ECM（FN; COL1; α-SMA）的mRNA和蛋白表达水平较Scramble组显著降低,P<0.05。而Transwell计数实验显示HES1 shRNA组HTMCs的迁移数量较Scramble组增加,P<0.05;CCK-8细胞活性分析实验表明抑制HES1表达对HTMCs的增殖活性影响不大,差异不具有统计学意义,P>0.05。 结论: 成功建立了稳定表达HES1 shRNA的HTMCs细胞株。HES1可以影响HTMCs中促纤维ECM的生成,及HTMCs的增殖和迁移能力。     

Glucocorticoids upregulate transepithelial electrical resistance and expression of tight junction-related protein in human trabecular meshwork cells

The trabecular meshwork is located at the anterior chamber angle, and is the main route for the outflow of aqueous humor. It is composed of perforated sheets of collagen and elastic tissue covered by trabecular meshwork (TM) cells, forming a filter with decreasing pore size as the canal of Schlemm is approached. TM cells have some endothelial properties, such as the presence of intercellular junctional complexes, particularly tight junctions (TJs). TJs form paracellular seals between adjacent cells and act as fences that segregate protein (and partially lipid) components of the apical and basolateral plasma membrane domains. Under the electron microscope, TJs appear as a series of discrete contacts between the lateral membranes of adjacent cells.     

生存素反义寡核苷酸对人子宫内膜癌细胞增殖的抑制作用

 目的 探讨生存素（survivin）反义寡核苷酸（antisense oligonucleotide,ASODN）对人子宫内膜癌细胞HEC-1B增殖的抑制作用。方法 实验分空白对照组（control组）、单纯脂质体对照组（Lip组）、正义链转染对照组（SODN组）、ASODN转染组（ASODN组）4组。人工合成正、反义寡核苷酸,经脂质体将survivin正、反义寡核苷酸转染入子宫内膜癌细胞48 h后收集各组细胞。免疫印迹（Western blot）法检测各组细胞生存素表达情况,流式细胞仪检测各组细胞凋亡率,四甲基偶氮唑蓝试验（MTT）法检测细胞生长抑制情况。结果 脂质体介导生存素反义寡核苷酸转染后的子宫内膜癌细胞出现生存素蛋白表达明显下降;ASODN转染组细胞凋亡率和增殖抑制率均明显高于各对照组（P<0.05）,而各对照组间差异无统计学意义（P>0.05）。结论 生存素反义寡核苷酸转染子宫内膜癌细胞能下调生存素蛋白表达,诱导子宫内膜癌细胞凋亡,抑制细胞增殖,对子宫内膜癌是一种有效的基因疗法。     

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。     

Wilms肿瘤基因反义寡苷酸对白血病细胞凋亡的影响

目的 了解Ｗｉｌｍｓ肿瘤基因（ＷＴ１）反义寡核苷酸（ＡＳＯ）对白血病细胞凋亡的作用。方法 应用ＷＴ１ＡＳＯ以及足叶乙甙（ＶＰ－１６）作用Ｋ５６２、ＨＬ－６０细胞系,然后应用流式细胞仪测定ＤＮＡ含量以确定白血病细胞的凋亡数。结果 Ｋ５６２细胞系经ＷＴ１ ＡＳＯ作用２４ｈ和６０ｈ后细胞凋亡数分别为１４．６％和２６．８％;而ＷＴ１有义寡核苷酸（ＳＯ）组分别为３．１％（２４ｈ）和３．９％（６０ｈ）;加入少