表皮生长因子对牛眼小梁细胞c—fos基因表达的诱导

目的研究表皮生长因子（ｅｐｉｄｅｒｍａｌｇｒｏｗｔｈｆａｃｔｏｒ，ＥＧＦ）对小梁细胞的作用。方法应用分子杂交技术研究ＥＧＦ对体外培养的牛眼小梁细胞ｃ－ｆｏｓ基因表达的诱导和３Ｈ胸腺核苷酸（３Ｈ－ｔｈｙｍｉｄｉｎｅｉｎｃｏｒｐａｒａｔｉｏｎ，３Ｈ－ＴｄＲ）掺入法观察细胞ＤＮＡ的合成。结果小梁细胞的３Ｈ－ＴｄＲ掺入率随着ＥＧＦ浓度不同而变化，浓度为２０～１５０ｎｇ／ｍｌ时，细胞掺入率随浓度增加升高（Ｐ＜０．０１）。与对照组相比，ＥＧＦ刺激停止生长的小梁细胞０．５小时后，ｃ－ｆｏｓ基因开始表达，１小时后达高峰，至２小时后消失。不同浓度ＥＧＦ刺激小梁细胞１小时后，ｃ－ｆｏｓ基因表达呈浓度依赖性。结论实验结果表明ＥＧＦ刺激小梁细胞增殖或进入生长状态，可能与ｃ－ｆｏｓ基因产物的信号传递作用有关。     

纤维连接蛋白对原发性开角型青光眼小梁网细胞增殖、黏附和迁移的影响

目的通过研究纤维连接蛋白（FN）对体外培养的原发性开角型青光眼（POAG）患者小梁网细胞增殖、黏附、迁移的影响,探讨FN与POAG发病机制的关系。方法取临床确诊的POAG患者小梁网切除术中的深层巩膜组织块,进行小梁网细胞体外原代和传代培养．并对其进行免疫细胞化学和电镜鉴定。取第3代小梁网细胞,实验组分别加入浓度为5、10、20、40、100μg／ml的FN（含无血清DMEM／F12培养液）培养,对照组不加FN。培养24h后,采用CCK-8比色法和Transwell试剂盒检测各组光密度值,分析FN对POAG小梁网细胞增殖、黏附、迁移的影响。采用SPSS17．0统计软件,组间比较采用One-Way ANOVA分析,两两比较采用SNK检验。结果经过免疫细胞化学和电镜鉴定后,确定培养的细胞为POAG小梁网细胞。不同浓度FN对小梁网细胞均有影响。FN对POAG小梁网细胞有促增殖作用,在FN为5～10μg／ml浓度范围,小梁网细胞增殖相对缓慢,10μg／ml以后呈现上调趋势,在40μg／ml时达到增殖高峰,100μg／ml仍促进增殖,但是相对缓慢。各实验组光密度值分别与阴性对照组比较,差异均有统计学意义（P〈0．01）;各实验组不同浓度组间比较,差异有统计学意义（F=81．778,P〈0．05）。FN浓度为5、10、20、40、100μg／ml时．小梁网细胞的增殖率分别为18．6％、54．7％、67．9％、98．7％和121．5％。同样,FN对POAG小梁网细胞有明显的促黏附、迁移作用。小梁网细胞的黏附、迁移能力随FN浓度的增加而增强,基本呈现曲线上升趋势,与阴性对照组比较,差异均有统计学意义（P〈0．05）。结论FN能促进POAG患者小梁网细胞增殖、黏附和迁移,其在POAG发病机制中的作用可能为通过影响POAG小梁网细胞的增殖、黏附、迁移功能参与房水流出的调节过程。     

柔毛霉素对兔晶体上皮细胞体外生长抑制的实验研究

目的研究柔毛霉素（daunomycin）对兔晶体上皮细胞体外生长抑制．探索使用柔毛霉素以预防后发障的发生。方法分离培养家兔晶体上皮细胞，传代后在24孔培养板培养72小时，计数后加入不同浓度的柔毛霉素作用10分钟后．吸出药物并冲洗后培养5天。结果柔毛霉素的LD50为0．52μg／ml～0．77μg／ml．经高浓度药物（10μp／ml）作用后的标本几乎无细胞存活，经0．5μg／ml药物浓度作用后的细胞形态发生变化。结论显示出低浓度柔毛霉素短时间作用后即能有效抑制晶体上皮细胞的增生，尤其便于术中一次性灌注用药，在降低后发障的发生具有较好的作用。     

转移生长因子-β2介导的小梁细胞纤维联结蛋白的mRNA及蛋白质的表达

目的探讨转移生长因子-β2（TGF—β2）对猪眼小梁细胞中纤维联结蛋白的mRNA及蛋白质表达的影响。方法经器官培养法（organ culture）2～3天后，分组的猪眼前段分别注入浓度为0．45ng／ml（原发性开角型青光眼前房水中TGF—β2的浓度）和0．18ng／ml（正常人眼前房水中TGF—β2的浓度）的活性TGF—β2，对照眼灌注不含TGF—β2的培养基，将猪眼前节经固定、石蜡包埋和切片，通过原位杂交和免疫组化的方法检测小梁网细胞中纤维联结蛋白的mRNA和蛋白质的表达，对照组采用同样的方法检测。结果在灌注含有0．45ng／ml活性TGF—β2的标本中，小梁细胞中纤维联结蛋白的杂交信号和免疫反应强度均呈强阳性，而在灌注含有0．18ng／ml TGF—β2的标本中，小梁细胞其杂交信号和免疫反应强度均较弱，在对照组中，杂交信号和免疫组化染色呈阴性。结论TGF—β2对灌注猪眼中小梁网的纤维联结蛋白的mRNA和蛋白质表达起正调节作用，表明猪眼小梁细胞合成的这种ECM（细胞外基质成分，即纤维联结蛋白）被房水中的TGF—β2调节，这个结论和我们以及其他研究者先前的假设是一致的，即浓度升高的活化TGF—β2可能在猪眼的小梁组织发生病理组织学改变中起着重要的作用。     

NF-κB对人眼小梁细胞增生及DNA合成的影响

目的 研究核转录因子-kappaB（NF-κB）对人眼小梁细胞增生及DNA合成的影响,探讨继发性青光眼的发病机制。方法 原代培养人眼小梁细胞,通过脂质体转染的方法,将P65表达质粒转染小梁细胞,24h后分别通过MTT法及3H-TdR掺入实验检测小梁细胞的增生及DNA合成情况。结果 小梁细胞P65质粒转染后细胞增生及DNA的合成均明显受抑制,与对照组相比差异有统计学意义（P〈0.05）。结论 P65的高表达抑制小梁细胞的增生及DNA的合成。     

白细胞介素-1β对人眼小梁细胞基质金属蛋白酶-3表达的影响

目的观察自细胞介素-1β（IL-1β）对体外培养的人眼小梁细胞基质金属蛋白酶-3（matrix metalloproteinases 3，MMP-3）表达的影响。方法采用组织块培养法对人眼小梁细胞（human trabecular ceils，HTCs）进行体外原代及传代培养，取传3代小梁细胞分别加入含有IL-1β终浓度为0ng／ml（对照组）、5、10、25、50ng／ml的无血清培养液，24h后采用RT-PCR法及ELISA法检测MMP-3量的变化。结果RT-PCR法检测IL-1β终浓度为5、10、25、50ng／ml实验组MMP-3／GAPDH的相对灰度比值分别为0．3437±0．0764、0．8588±0．0443、1．0079±0．0347、1．3466±0．0309，均高于对照组的相对灰度比值0．1037±0．0494，且各实验组与对照组相比差异有统计学意义（P〈0．05）；ELISA法检测IL-1β终浓度为5、10、25、50ng／ml实验组的MMP-33浓度值分别为（2．0325±0．2456）ng／ml、（2．4607±0．350）ng／ml、（2．8532±0．1392）ng／ml、（3．5858±0．1141）ng／ml，均高于对照组的浓度值（1．2437±0．3910）ng／ml，且各实验组与对照组相比差异有统计学意义（P〈0．05）。结论IL-1β在一定范围内促进MMP-3的表达，并且呈现一定的剂量依赖性，推测IL-1β可以通过促进MMP-3的表达来减少小梁网组织细胞外基质（ECM）的堆积，使房水引流通畅，降低眼内压。     

肿瘤坏死因子-α对体外培养的人眼小梁细胞基质金属蛋白酶-3和基层金属蛋白酶-9表达的影响

目的 探讨肿瘤坏死因子-α，（tumor necrosis factor-α．TNF—α）对体外培养的人眼小梁细胞基质金属蛋白酶（matrix metalloproteinases，MMP-3、MMP-9）表达的影响。方法 对人眼小梁细胞（humantrabecular cells，HTCs）进行体外原代培养及传代培养。取传3代的小梁细胞分别施加含TNF-α终浓度为0（对照组）、1mg／ml、10mg／ml、25ng／ml的无血清培养液．48h后采用免疫细胞化学法观察小梁细胞MMP-3、MMP-9的染色变化。对结果进行计算机图像分析并进行统计学检验．结果 利用免疫细胞化学法检测1ng／ml、10ng／ml、25ng／ml的TNF-α实验组小梁细胞MMP-3的平均灰度值分别为138．63±8．15、138．73±7．02、129.25±10．47．均低于对照组平均灰度值143．53±6．01．且各实验组与对照组比较差异均有显著性（P〈0．05）：实验组小梁细胞MMP-9的平均灰度值分别为137．60±11．53、125．20±17．29、119．88±12．47．均低于对照组平均灰度值145．30±7．05．且各实验组与对照组比较差异均有显著性（P〈0．05）。结论 TNF-α在-定范围内促进小梁细胞MMP-3及MMP-9的表达．增加了小梁网异常堆积的细胞外基质（extracellular matrix，ECM）的降解，使房水流出增加，眼压降低。     

灯盏花素对体外培养牛小梁细胞及细胞外基质的影响

目的：探讨灯盏花素是否具有促进体外培养牛小梁细胞增殖并降低其分泌细胞外基质的作用。方法：在体外培养的第三代牛小梁细胞中加入中药灯盏花的提取物灯盏花素，培养24h后，采用放射免疫分析法测定培养液中透明质酸（hyaluronic acid,HA)，层粘连蛋白（laminin,LN），胶原蛋白Ⅲ型（collagen-Ⅲ，PC-Ⅲ）的含量，同时采用噻唑蓝（MTT）比色法测定活细胞的光密度值从而反映活细胞的数量。结果：5mg/ml,0.5mg/ml浓度的灯盏花素组，牛小梁细胞数量增多；5mg/ml,0.5mg/ml浓度的灯盏花素组，培养液中胶原蛋白Ⅲ型的浓度低降低，0.05mg/ml浓度的灯盏花素组，透明质酸和层粘连蛋白的浓度降低；硼酸缓冲液对细胞增殖及细胞基质浓度均无影响。结论：灯盏花素具有促进牛小梁细胞增殖，降低牛小梁细胞分泌细胞外基质（透明质酸、胶原蛋白Ⅲ型和层粘连蛋白）的作用。     

雷帕霉素对兔晶状体上皮细胞增殖的影响

目的研究雷帕霉素（rapamycin）对培养的兔晶状体上皮细胞增殖的作用及其对细胞周期的影响。方法将传代的兔晶状体上皮细胞用含不同雷帕霉素浓度的培养液培养24h后，用NIT法测定5ng／ml、10ng／ml、20ng／ml、40ng/ml、80ng/ml雷帕霉素浓度组及对照组细胞的增殖情况；用流式细胞仪测定20ng/ml雷帕霉素浓度组及对照组细胞周期的变化情况。结果用5ng/ml的培养液培养细胞时，细胞增殖受到抑制（P〈0．05），随药物浓度增加，细胞增殖受抑制越明显；用10ng/ml的培养液培养细胞时，细胞增殖明显受到抑制（P〈0．01）。细胞周期分析显示，雷帕霉素（20ng/ml）作用后，G0／G1期细胞较对照组增多（由51．72％增加到61．63％），S期细胞较对照组减少（由34．92％减少至10．85％）。结论雷帕霉素具有抑制兔晶状体上皮细胞增殖的作用，且随浓度增加抑制作用增强。雷帕霉素将细胞抑制在G1期的限制点内。     

NF-κB活性对TNF-α调节小梁细胞表达MMP-3和MMP-9的影响

目的探讨核因子-κB（nuclear factor-κB，NF-κB）是否参与肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）调节人眼小梁细胞（human trabecular cells，HTCs）表达基质金属蛋白酶-3（matrix metalloproteinases-3，MMP-3）和基质 金属蛋白酶-9（matrixmetalloproteinases-9，MMP-9）的过程。方法采用RT-PCR法和酶谱分析法（zymography）检测体外培养的人眼小梁细胞经0ng／ml（对照组）、lng／ml、10ng／ml、25ng/ml TNF-α处理24h后，细胞表达MMP-3、MMP-9的量；运用NF-κB的特异抑制剂二硫氨基甲酸吡啶（pyrrolidine dithocarba-mate，PDTC）抑制NF-κB的活化，观察TNF-α对体外培养的HTCsMMP-3，MMP-9表达影响的改变。结果运用RT-PCR法和酶谱分析法研究均发现经浓度为1ng／ml、10ng／ml、25ng／ml的TNF-α处理的细胞在mRNA和上清液中活性蛋白水平均能增加MMP-3、MMP-9的表达，各组mRNA／β-actin吸光度比值和条带酶解量与对照组相比差异均有显著性（P〈0．05）。提前30min加入PDTC，MMP-3、MMP-9的表达量分别较未加入PDTC组表达量明显减少．差异有显著性（P〈0．05）。结论TNF-α可上调MMP-3及MMP-9的表达。PDTC能够下调TNF-α对小梁细胞MMP-3及MMP-9的表达，因此推测NF-κB可能参与MMP-3和MMP-9转录的启动．     

Effect of heparin on human corneal fibroblast proliferation in vitro with and without growth factor stimulation

· Background: The outcome of keratorefractive procedures such as PRK and LASIK is limited by the wound-healing process in the corneal stroma, which gives rise to complications such as haze formation and regression. The proliferation and matrix synthesis of corneal stromal fibroblasts is the central element of the wound-healing process. In order to develop new therapeutic strategies to reduce wound-healing intensity, we investigated the effect of heparin on the proliferation of cultured human corneal stromal fibroblasts (HCF) alone and in the presence of growth factors. · Methods: Primary cultures of HCF were established using epithelium and endothelium-free explants. Secondary cultures of HCF (first passage), cultured in WM/F12 supplemented with 10 μg/ml transferrin and 10 μg/ml thyroglobulin (LR-1 medium), 1% fetal calf serum (FCS) and 10% FCS were used to determine the effect of heparin on the proliferation of HCF in concentrations ranging from 12.5 μg/ml to 5000 μg/ml. Cell number was determined using the CASY 1 cell counter system. Modulation of HCF proliferation by heparin (50 μg/ml and 2000 μg/ml) was also investigated under serum-free conditions and in the presence of bFGF, EGF and PDGF-BB. · Results: Addition of heparin led to a dose-dependent inhibition of proliferation after 6 days of incubation, which was statistically significant for 500–5000 μg heparin/ml (FCS 1%) and for 200–5000 μg heparin/ml (FCS 10%). IC₅₀ values for this effect were determined to be approximately 700 μg heparin/ml. When cultured under serum-free conditions (LR-1), a significant reduction of cell number was only observed with 5000 μg heparin/ml. There was no significant modulation of PDGF-BB-, bFGF-, or EGF-stimulated cell proliferation by heparin at concentrations of 50 μg/ml and 2000 μg/ml after 6 days of incubation. · Conclusion: Our observations indicate that heparin can inhibit proliferation of HCF effectively. The results of the present study could eventually pave the way to prevent anterior stromal haze formation and regression after keratorefractive surgery. Received: 23 April 1998 Revised version received: 12 August 1998 Accepted: 13 August 1998     

γ—干扰素及肝素抑制兔晶体上皮细胞生长的实验研究

目的:研究γ—干扰素及肝素对体外培养的兔晶体上皮细胞(RLEC)的生长抑制作用,探讨后发性白内障的防治方法。方法:RLEC原代细胞来自正常健康的新西兰大白兔。胰酶消化法传代。第三代细胞分别与0.1～10000u/ml剂量的γ—干扰素及100～5000u/ml的肝素孵育48小时,用MTT法检测γ—干扰素及肝素对晶体上皮细胞生长的抑制作用效果。结果:0.1～100u/ml剂量组的γ—干扰素对体外培养的RLEC无明显的抑制作用,1000u/ml及10000u/ml剂量组的γ—干扰素则对RLEC有较明显的抑制作用,其增殖抑制率分别为27％及38％。100～5000u/ml剂量组的肝素对RLEC均有明显的抑制作用。结论:一定浓度的γ—干扰素及肝素对兔晶体上皮细胞生长具有直接抑制作用。眼科学报1997;13:167—169。     

Prostaglandin production by human trabecular cells: in vitro inhibition by dexamethasone

In addition to the well-known ability of prostaglandins (PGs) to raise intraocular pressure (IOP), it recently has been reported that moderate and low doses of PGE2 and PGF2 alpha significantly reduce IOP in a variety of experimental animals. These studies suggested to us that PGs might serve as endogenous regulators of outflow facility in the meshwork if these autacoids were produced and secreted by human trabecular cells. To examine this possibility, media from well-defined trabecular cell material were assayed using specific radioimmunoassays. Morphologically differentiated human trabecular cells produced high levels of PGE2, and somewhat lower levels of PGF2 alpha and 6KF1 alpha in the presence and absence of serum. In a typical experiment, the following PG levels were detected in the cell culture media after 24 hours: PGE2; 225; PGF2 alpha, 33.5; 6KF1 alpha, 12.7 ng/ml with the presence of 10% fetal calf serum; and PGE2, 30.0; PGF2 alpha, 4.8; 6KF1 alpha, 3.6 ng/ml in serum-free media. Since glucocorticoids are known to inhibit PG pathways in other tissues, this effect was examined in the cultured trabecular cells. Moderate concentrations of dexamethasone (DEX) produced a marked inhibition in the levels of all three PGs. For PGE2 production, 10(-8) M DEX inhibited approximately 75%, and 10(-7) M DEX inhibited approximately 90%. More detailed dose-response studies revealed that the I50 for inhibition of PG production by dexamethasone was less than 10 nM, thus indicating that the steroid effect probably involved high affinity glucocorticoid receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulation of myocilin/TIGR expression in human trabecular meshwork.

PURPOSE: To study factors that modulate myocilin/trabecular meshwork inducible glucocorticoid response protein (TIGR) mRNA expression in human trabecular meshwork (TM). METHODS: mRNA from fresh TM of four human donors, from perfused anterior segment organ cultured TM of three donors, and from four primary TM cell lines of different donors was isolated. The full length cDNA of myocilin/TIGR was cloned from TM mRNA using a polymerase chain reaction approach and used as probe for northern blot analysis hybridization. Trabecular meshwork cell cultures were treated with transforming growth factor (TGF)-beta1 (1 ng/ml), dexamethasone (10(-7) M), and mechanical stretch (10%). RESULTS: mRNA for myocilin/TIGR could be readily detected by northern blot analysis hybridization in 2 to 3 microg of total RNA from all fresh and all organ-cultured TM samples. In contrast, no mRNA for myocilin/TIGR could be detected in 20 microg of total RNA isolated from three different primary TM cell lines. Only one TM cell line had a baseline expression of myocilin/TIGR, which was 35- to 55-fold lower than that of fresh or organ-cultured TM samples. Treatment of TM cell cultures with dexamethasone for 1 day markedly increased expression of myocilin/TIGR mRNA, an effect that was even more pronounced after 3 days of treatment. Treatment with TGF-beta1 for 24 hours had no effect; however, after 3 and 12 days of treatment a 3.8- and 4-fold increase in myocilin/TIGR mRNA expression was observed. Expression of myocilin/TIGR mRNA was also increased after 10% mechanical stretch; however, in contrast to the effects of TGF-beta-1, this effect was observed much earlier (8-24 hours) after treatment. CONCLUSIONS: Dynamic mechanical stimuli maintain myocilin/TIGR expression in TM in situ and lack of these stimuli in monolayer cell cultures might be involved in downregulation of myocilin/TIGR expression.     

The effect of growth factors on the proliferation of cultured porcine trabecular meshwork cells

PURPOSE: We attempted to identify cell growth factors that cause a multiplication of trabecular meshwork(TM) cells. METHODS: Porcine TM cells were cultured in Dulbecco's modified Eagle's medium/nutrient mixture F-12/Ham to which we added 1, 10, and 100 ng/ml of platelet-derived growth factor(PDGF), fibroblast growth factor 2(FGF2), insulin-like growth factor-1 (IGF-1), vascular endothelial cell growth factor(VEGF), hepatocyte growth factor (HGF), or brain-derived neurotrophic factor(BDNF). We measured [3H] thymidine incorporation to evaluate the influence of the growth factors on TM cell proliferation. RESULTS: [3H] thymidine incorporation into TM cells was promoted by 10 and 100 ng/ml of PDGF, IGF-1, and FGF2 after 24 and 48 hours, whereas 1, 10, and 100 ng/ml of VEGF restrained cell proliferation after 48 hours. HGF and BDNF did not show any remarkable influence on TM cell proliferation. CONCLUSIONS: Our, results suggest that PDGF, IGF-1, and FGF2 may cause a drop in intraocular pressure followed by activation of a TM function, by multiplying TM cells.     

Effects of an anti-prostaglandin agent added to the irrigation solution on damage to the anterior segment in monkey eyes induced by pars plana vitrectomy

Flurbiprofen is a water-soluble, non-steroid anti-inflammatory agent and is capable of marked inhibition of prostaglandin synthesis. In a previous study by the authors, it was determined that the optimum concentration of flurbiprofen for intraocular irrigation solution in monkey retinas is 4 to 40 micrograms/ml. The present study was conducted to determine the optimum concentration of flurbiprofen for the irrigation solution, as well as to evaluate its toxic effects on the anterior segment tissues. In a pilot experiment, flurbiprofen was injected directly into the anterior chamber of pigmented Japanese rabbits to investigate its overall toxicity and to quantify the concentration at which it could become toxic to the cornea. This experiment showed that aqueous concentrations of flurbiprofen greater than 1,000 micrograms/ml were toxic. In a subsequent study, the twelve eyes of six Japanese monkeys (Macaca fuscata) underwent vitrectomy using balanced salt solution (BSS) containing concentrations of flurbiprofen from 4 to 800 micrograms/ml and BSS alone. This experiment showed that BSS containing 4 to 100 micrograms/ml of flurbiprofen prevented post-operative occurrence of edema in non-pigmented epithelial cells in the ciliary body. Furthermore, it was observed that infiltration of inflammatory cells into the trabecular meshwork and swollen mitochondria in the corneal endothelial cells were likewise prevented. This is thought to be due to the pharmacological action of flurbiprofen; which has an inhibitory effect on leucocyte migration and prostaglandin synthesis as well as stabilization of the cell membrane. However, these effects were not observed in the eyes vitrectomized with flurbiprofen concentrations between 400 and 800 micrograms/ml.(ABSTRACT TRUNCATED AT 250 WORDS)