小檗胺抑制兔Tenon囊和滤过道内成纤维细胞增殖的实验研究

目的:研究钙调素拮抗剂小檗胺(berbamine,BER)对兔Tenon囊成纤维细胞和兔小梁切除术后滤过道内成纤维细胞增殖的抑制作用。方法:体外培养兔Tenon囊成纤维细胞,经不同浓度BER处理不同时间后,细胞计数Kit8(CCK8)法检测BER对兔Tenon囊成纤维细胞增殖的抑制作用,流式细胞仪检测其凋亡率及细胞周期的变化。HE染色检测BER对兔小梁切除术后滤过道内成纤维细胞增殖的抑制作用。结果:兔Tenon囊成纤维细胞经不同浓度BER处理不同时间后细胞增殖受到抑制,并呈时间剂量依赖性。流式细胞仪检测发现BER处理后兔Tenon囊成纤维细胞呈现G1期阻滞,细胞凋亡率明显增加,20mg/LBER处理9h,细胞凋亡率从0.64%增加到31.86%。HE染色显示BER显著抑制兔小梁切除术后滤过道内成纤维细胞的增殖。结论:小檗胺在体内外均能抑制成纤维细胞的增殖,其可能是通过诱导细胞凋亡方式抑制兔Tenon囊和滤过道内成纤维细胞的增殖。     

汉防己甲素对体外人眼Tenon囊成纤维细胞的抑制效应

目的:研究汉防己甲素(tetrandrine,Tet)对人眼Tenon囊成纤维细胞(Tenon’s capsule fibroblasts,TCFs)的抑制效应。方法:用组织块和混合消化液培养法体外培养人眼Tenon囊成纤维细胞,并对培养的传代细胞进行形态学的鉴定。采用MTT法检测不同浓度的汉防己甲素(10-4,10-5,10-6,10-7mol/L)及0.2g/L丝裂霉素(MMC)对人眼Tenon囊成纤维细胞的抑制作用。结果:人眼Tenon成纤维细胞体外生长良好,经光镜和免疫组化观察证明该细胞为成纤维细胞。MTT法证实,随着Tet药物浓度的增大,TCFs增生活性降低,对应的TCFs抑制率升高。结论:Tet对人眼Tenon囊成纤维细胞的增殖具有明显的抑制作用。     

全反式维甲酸对翼状胬肉成纤维细胞增殖的影响

目的:观察全反式维甲酸(ATRA)对体外培养人翼状胬肉成纤维细胞(HPF)增殖的影响,寻找治疗和预防翼状胬肉复发的新途径。方法:体外培养翼状胬肉患者成纤维细胞;将不同浓度的ATRA作用于成纤维细胞分别达24,48h和72h后,MTT法检测ATRA对HPF的影响,免疫组织化学染色增生细胞核抗原(PCNA)检测细胞生长活性。结果:MTT法显示10-8mmol/LATRA作用24h后能抑制HPF的增殖(P<0.05),呈剂量和时间依赖性。ATRA能浓度依赖性地抑制细胞表达PCNA(P<0.05)。结论:ATRA能够显著抑制HPF的增殖。     

曲尼司特对青光眼患者体外培养的眼部Tenon囊成纤维细胞增殖和移行的影响

目的 探讨曲尼司特(tranilast)对青光眼患者体外培养的眼部Tenon囊成纤维细胞增殖及移行的影响。方法 取青光眼患者滤过术中剪下的Tenon囊组织,在体外进行成纤维细胞原代及传代培养。分别采用MTT法、细胞计数法、免疫组化加图像分析法及划线法,研究不同浓度的曲尼司特对体外培养的成纤维细胞增殖及移行的影响及其与蛋白激酶C(PKC)表达的关系。结果 当浓度在12．5～100．0mg／L之间变化时,曲尼司特能明显抑制成纤维细胞的增殖,强度呈剂量依赖性;50．0mg／L和100．0mg／L的曲尼司特能明显抑制成纤维细胞的移行,并下调细胞内PKC的表达。结论 曲尼司特能抑制成纤维细胞的增殖及移行,这种作用可能与下调细胞内PKC的表达有关。     

蛇毒制剂对体外培养的兔结膜下成纤维细胞的影响

目的　观察蛇毒制剂对体外培养的兔结膜下成纤维细胞与胶原黏附及细胞移行和增殖的影响。方法　将培养的第3～5代兔结膜下成纤维细胞于不同浓度的蛇毒制剂孵育后,接种于涂有鼠尾胶原的24孔板中,去除未黏附的细胞,用目镜网格器计数法和四甲基偶氮唑盐法记录贴壁细胞量。在第3代生长融合细胞的培养板上划线,造成无细胞的裸露区,在不同浓度的蛇毒制剂下孵育,用目镜网格器每6h观察记录移行至裸露区的细胞数。第3代兔结膜下成纤维细胞接种于24孔板与不同浓度的蛇毒制剂共育24、48h后,用四甲基偶氮唑盐法记录各孔细胞数量的吸光度(A)值。结果蛇毒制剂抑制结膜下成纤维细胞黏附呈剂量依赖性。抑制成纤维细胞黏附的半数有效量(ID50)为1.0×10     

苏拉明抑制体外培养的Tenon囊成纤维细胞增殖的实验研究

目的观察苏拉明对体外培养的Tenon囊成纤维细胞增殖的影响,探讨其作为青光眼滤过术辅助用药的可能性。方法通过体外培养青光眼患者Tenon囊成纤维细胞,采用四甲基偶氮唑盐(MTT)比色法检测不同作用时间及不同浓度的苏拉明对细胞增殖的抑制作用,并用流式细胞术分析细胞周期。结果MTT法显示苏拉明作用24h、48h及72h后,浓度分别为500mg·L-1、400mg·L-1、300mg·L-1以上的各实验组与对照组吸光度值A均数间比较有显著性差异;流式细胞术显示苏拉明将细胞阻抑于G2/M期,实验组G2/M期的细胞比对照组增加了15.8%.结论苏拉明能抑制体外培养的Tenon囊成纤维细胞增殖,并呈现剂量、时间依赖性,提示其有望成为青光眼滤过术后抗瘢痕增生的辅助用药。     

姜黄素抑制翼状胬肉成纤维细胞增生的研究

目的观察姜黄素对体外培养人翼状胬肉成纤维细胞(HPF)增生和凋亡的影响,寻找辅助治疗和预防翼状胬肉复发的新方法。方法用0～160μmol/L姜黄素作用体外培养的HPF,24～96h后观察不同浓度姜黄素对HPF的影响。MTT法及免疫组织化学染色增生细胞核抗原(PCNA)检测细胞生长活性,流式细胞仪测定细胞周期时相变化。结果在20～160μmol/L浓度作用24～72h范围内,姜黄素可剂量和时间依赖性的抑制HPF增生(P<0.05)。20,40,80,160μmol/L姜黄素作用24h后,流式细胞仪检测结果发现,姜黄素作用组均出现典型的亚二倍体凋亡峰,G0/G1期细胞百分比上升(P<0.05),S期细胞百分比下降(P<0.05),说明细胞阻滞于G0/G1期。当姜黄素的浓度在20～160μmol/L范围内能浓度依赖性地抑制细胞表达PCNA(P<0.05)。结论姜黄素可显著抑制翼状胬肉成纤维细胞增生,使细胞周期停滞于G0/G1期,并诱导其凋亡,作用呈时间和剂量依赖性。     

曲古抑菌素A对人Tenon囊成纤维细胞作用的研究

目的：研究曲古抑菌素A( trichostatin A,TSA)对体外培养的人Tenon囊成纤维细胞( human Tenon capsule fibroblast, HTF)增殖能力以及组蛋白去乙酰化酶1( histone deacetylase 1,HDAC1)和HDAC2蛋白表达的影响。
　　方法：取青光眼滤过术中Tenon囊组织进行HTF体外培养。选取第3~6代细胞进行实验。设置空白对照组及TSA组(600nmol/L TSA加入培养液中),分别于培养后1,2,3 d应用 MTT法检测细胞活力变化;以600 nmol/L TSA处理 HTF 2 d 后, Western blot 法检测细胞中 HDAC1和HDAC2蛋白的表达。
　　结果：与对照组比较,TSA作用1d后HTF活力下降,呈时间依赖性,具有统计学差异( P<0.05)。 TSA处理HTF后2d,Western blot法检测HDAC1和HDAC2蛋白表达受到明显抑制。
　　结论：TSA可以通过抑制HDAC1和HDAC2的表达量,抑制人Tenon囊成纤维细胞增殖,从而减少术后结膜瘢痕形成可能。     

NF -κB抑制剂吡咯烷二硫氨基甲酸对人翼状胬肉成纤维细胞增殖的影响

目的:研究(nuclear factor,NF)2κB抑制剂吡咯烷二硫氨基甲酸(NF-B Inhibitor pyrrolidine dithiocarbamate,PDTC)对体外培养人翼状胬肉成纤维细胞(human pterygium fibroblast,HPF)增殖的影响,寻找辅助治疗和预防翼状胬肉复发的新方法.方法:取人翼状胬肉标本进行体外贴壁细胞常规培养,取第3～6代细胞进行试验.(1)免疫组化法染色结合细胞生物学特征鉴定成纤维细胞;(2)不同浓度PDTC(2.5,5,10,20,40 μmol/L)干预成纤维细胞24,48,72 h后,MTT法检测细胞生长抑制率;(3)免疫细胞化学法分别检测PDTC干预48 h后HPF增殖细胞核抗原(PCNA)的表达情况.结果:体外培养的翼状胬肉细胞经免疫组化染色可见波形蛋白表达阳性,结合细胞生物学特性可以确定为成纤维细胞.MTT实验表明,随着PDTC干预浓度的增大和时间延长,成纤维细胞生长抑制率增加(P＜0.05),在2.5-40 μmol/L浓度作用24-72 h,PDTC可呈剂量和时间依赖性.PDTC干预后PCNA蛋白表达均下降.当PDTC的浓度在5,10,20,40 μmol/L范围内能浓度依赖性地抑制细胞表达PCNA(P＜0.05).结论:PDTC对体外培养的HPF细胞有抑制作用,在一定浓度和时间范围内抑制作用呈剂量与时间依赖性.     

粉防己碱对翼状胬肉成纤维细胞增生作用的研究(英文)

目的:观察粉防己碱(tetrandrine,Tet)对体外培养人翼状胬肉成纤维细胞(human pterygium fibroblast,HPF)增殖的影响,研究粉防己碱对翼状胬肉成纤维细胞增殖的作用,寻找辅助治疗翼状胬肉的新方法。方法:用不同浓度(0 ～160μmol/L)Tet作用体外培养的HPF,观察24 ～96h Tet对HPF的影响。MTT法检测细胞生长抑制率,免疫细胞化学法检测Tet干预前后HPF增殖细胞核抗原(PCNA)的表达情况。结果:在Tet20, 40, 80, 160μmol/L浓度作用24 ～72h范围内,可剂量和时间依赖性的抑制HPF增生(P<0.05)。Tet干预后PCNA蛋白表达下降,当Tet的浓度在20 ～160μmol/L范围内能浓度依赖性地抑制细胞表达PCNA(P<0.05)结论:Tet可显著抑制翼状胬肉成纤维细胞增生,在一定浓度和时间范围内抑制作用呈剂量与时间依赖性。但在高浓度时( >160μmol/L)可能存在细胞毒性作用。     

Interferon-gamma inhibits collagen synthesis by human Tenon's capsule fibroblasts in vitro

Collagen deposition is largely responsible for scar information, an undesirable outcome of glaucoma filtering surgery. Because interferon-gamma (IFN-gamma) has been reported to inhibit collagen synthesis in a variety of cells, the authors examined its effect on collagen synthesis by Tenon's capsule fibroblasts cultured from three patients. In addition, the authors studied the effects of IFN-gamma on fibroblast proliferation. IFN-gamma inhibited collagen synthesis at dosages of 1-10,000 U/ml. The average median effective dose (ED50) was 6.60 U/ml. In contrast, IFN-gamma had no significant effect on cell proliferation in dosages of 0.001-10,000 U/ml. These results suggest that IFN-gamma may be a useful agent for modifying the wound healing response, particularly after glaucoma filtering surgery.     

精氨酸-甘氨酸-天门冬氨酸-丝氨酸肽对人Tenon囊成纤维细胞的贴壁抑制试验观察

目的：研究精氨酸-甘氨酸-天门冬氨酸-丝氨酸（Arg-Gly-Asp-Ser，RGDS）肽对人Tenon囊成纤维细胞与纤维连接蛋白（FN）粘附、增殖的影响。方法：在传代培养的人Tenon囊成纤维细胞中加入不同浓度的RGDS（1、0.1、0.001g/L），用未贴壁细胞记数法及MTT法测定RGDS肽对人Tenon囊成纤维细胞粘附、增殖的影响。结果：RGDS肽能抑制成纤维细胞在FN上的粘附，呈浓度效应特点，能抑制成纤维细胞的增殖，呈浓度及时间效应特点。结论：在细胞水平上证实RGDS肽可阻止人Tenon囊成纤维细胞对FN的粘附和增殖，具有避免青光眼滤过术后瘢痕化形成的应用前景。     

Anti-allergic drugs inhibit the proliferation of human Tenon's capsule fibroblast and maintain the experimental filtering blebs on rabbit eyes

PURPOSE: To examine whether tranilast and ketotifen fumarate inhibit the growth of human Tenon's capsule fibroblasts and maintain experimental filtering blebs on rabbit eyes. METHODS: Human Tenon's capsule fibroblasts were grown in Dulbecco's modified Eagle's medium with 10% fetal calf serum and growth was measured with 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and direct counting of cell number. Production of collagen and transforming growth factor-beta 1 (TGF-beta 1) was measured with corresponding enzyme-linked immunosorbent assay (ELISA). In experimental filtering surgery performed on rabbit eyes, the effects of the topical drugs on intraocular pressure and on maintenance of the filtering blebs were observed. RESULTS: Both tranilast and ketotifen inhibited the growth of the fibroblasts and half-inhibitory concentrations were 200 microM and 70 microM, respectively. Low concentration (10 microM) of tranilast and ketotifen decreased the synthesis of collagen but neither drug had any obvious effect on TGF-beta 1 production. Both drugs prolonged the life of the experimental filtering bleb significantly [12.5 +/- 3.2 (mean +/- standard deviation) days, 13.9 +/- 3.4 days, respectively; control, 10.5 +/- 2.4 days]. CONCLUSION: Tranilast and ketotifen inhibited the growth of human Tenon's capsule proliferation and are capable of maintaining experimental filtering blebs in rabbit eyes.     

汉防己甲素诱导人眼Tenon囊成纤维细胞的凋亡效应

目的 研究汉防己甲素(Tet)抑制人眼Tenon囊成纤维细胞(TCFs)的凋亡效应.方法 用组织块混合消化液培养法体外培养人眼TCFs,并对培养的传代细胞进行形态学鉴定.通过透射电镜、TUNEL法和流式细胞仪观察Tet抑制人眼TCFs的凋亡作用.结果 人眼TCFs体外生长良好.透射电镜下见Tet处理后的人眼TCFs细胞质固缩,呈现凋亡表现;TUNEL法可见Tet组出现大量的阳性凋亡细胞;流式细胞仪检测TCFs经Tet作用后G_0/G_1期细胞减少22.2%,S期细胞增加20.53%,G_2/M期细胞增加1.6%,Tet抑制TCFs细胞周期于G1期,Tet组凋亡率明显高于对照组(P=0.000).结论 Tet可以诱导人眼TCFs发生凋亡,进而发挥其抑制增生的作用.     

汉防己甲素抑制人眼Tenon囊成纤维细胞的凋亡效应

目的研究汉防己甲素（Tet）抑制人眼Tenon囊成纤维细胞（TCFs）的凋亡效应。方法用组织块混合消化液培养法体外培养人眼TCFs,并对培养的传代细胞进行形态学鉴定。通过透射电镜、TUNEL法和流式细胞仪观察Tet抑制人眼TCFs的凋亡作用。结果人眼TCFs体外生长良好。透射电镜下见Tet处理后的人眼TCFs细胞质固缩,呈现凋亡表现;TUNEL法可见Tet组出现大量的阳性凋亡细胞;流式细胞仪检测TCFs经Tet作用后G0／G1期细胞减少22．2％,S期细胞增加20．53％,G2／M期细胞增加1．6％,Tet抑制TCFs细胞周期于G,期,Tet组凋亡率明显高于对照组（P=0．000）。结论Tet可以诱导人眼TCFs发生凋亡,进而发挥其抑制增生的作用。     

Trifluorothymidine and 5-fluorouracil: antiproliferative activity in tissue culture

The antiproliferative activity of trifluorothymidine (F3TDR) and 5-fluorouracil (5-FU), two cell-cycle-phase-specific antimetabolites, was compared in a tissue culture model of human scleral fibroblasts and rabbit corneal epithelial cells. The mean concentrations required to inhibit human scleral fibroblast proliferation to 50% of the control rate (ID50) after 5 days of exposure were 8.50 micrograms/mL for F3TDR and 0.43 microgram/mL for 5-FU. The corresponding ID50 levels for rabbit corneal epithelial cells were 0.24 microgram/mL and 0.42 microgram/mL. The ID50 of F3TDR was significantly higher than that of 5-FU in human scleral fibroblasts (p less than 0.001) but not in rabbit corneal epithelial cells. F3TDR appears to have no clinical advantage over 5-FU in minimizing corneal epithelial toxicity when given to prevent fibroblast proliferation after glaucoma filtering surgery.     

Gamma-interferon effects on human fibroblasts from Tenon's capsule

Gamma-interferon (G-IFN) exhibits antiproliferative effects, inhibits collagen synthesis by fibroblasts, and may help to regulate the turnover of fibrous tissue. Fibroblasts from Tenon's capsule are cellular components that contribute to unsuccessful glaucoma filtration surgery. Therefore, the authors studied the effects of G-IFN on growth, wound closure, collagen synthesis, and extracellular matrix in cultured human Tenon's capsule fibroblasts (HTCF). HTCF incubated with G-IFN at doses of 1 to 1000 U/ml showed cellular heterogeneity, with some cells showing morphologic changes characteristic of senescence, and a dose-dependent inhibition of wound closure. At 1000 U/ml, G-IFN reduced collagen synthesis by 57%, reduced immunofluorescent staining of collagen type I and fibronectin network, and altered the organization of actin microfilaments into large cable-like structures. Cell proliferation and viability were not affected at any concentration of G-IFN. These results suggest that G-IFN may be useful to modulate wound healing after filtration surgery.     

Tranilast inhibits cell proliferation and collagen synthesis by rabbit corneal and Tenon's capsule fibroblasts

PURPOSE: To determine whether tranilast, N-(3,4-dimethoxycinnamoyl) anthranilic acid, influences cell proliferation and collagen synthesis by rabbit Tenon's capsule fibroblasts (TFs) and corneal stromal fibroblasts (CFs). METHODS: Rabbit TFs and CFs (7000 cells/well) were cultured in F-12 nutrient mixture supplemented with 1% FBS, plus 0, 3, 30, or 300 microM tranilast, and the number of cells was counted 72 hrs later. To determine the effect of tranilast on collagen synthesis, cells at confluence were cultured in a medium containing 0, 3, 30, or 300 microM tranilast and labeled with 3H-proline, and the amount of radioactivity incorporated into collagenase-sensitive proteins was measured. RESULTS: At 300 microM, tranilast decreased the number of TFs by about 27% and the number of CFs by about 45%, but had no effect on cell viability. The same concentration of tranilast reduced TFs collagen synthesis and CFs collagen synthesis. CONCLUSIONS: Tranilast may inhibit scar formation after trabeculectomy for glaucoma and after excimer laser photorefractive keratectomy.     

Effect of growth factors on the activation of human Tenon's capsule fibroblasts

PURPOSE: To investigate stimulatory effects of PDGF-AA, PDGF-AB, PDGF-BB, bFGF, IL-1beta, TGF-beta1 and TGF-beta2 on the proliferation and myofibroblast transformation of cultured human Tenon's capsule fibroblasts and to characterize expression of PDGF- and TGF-beta-receptors in these cells. METHODS: To determine cell proliferation, cell number of 2nd passage cultured human Tenon's capsule fibroblasts was measured before and after addition of growth factors using a computer-based cell counter system. Immunoblotting was used to detect and quantitate alpha-smooth-muscle actin (alpha-SMA) expression. Expression of PDGF- and TGF-beta-receptor mRNA was detected by RT-PCR, expression of the corresponding protein was demonstrated using Western blot. RESULTS: A significant increase in proliferation (p < or = 0.05) was detected after exogenous stimulation with PDGF-AA (10 ng/ml and 100 ng/ml), PDGF-AB (10 ng/ml and 100 ng/ml), PDGF-BB (10 ng/ml and 100 ng/ml), bFGF (100 ng/ml), IL-1beta (1 ng/ml and 10 ng/ml), TGF-beta1 (0.5 ng/ml) and TGF-beta2 (0.5 ng/ml). Both TGF-beta1 and TGF-beta2 stimulated expression of alpha-SMA in a dose dependent manner with peak activity at a concentration of 50 ng/ml (TGF-beta1) and 500 ng/ml (TGF-beta2). Protein and mRNA of PDGF-receptor type alpha and type beta and TGF-beta-receptors type I, II and III are expressed in cultured human Tenon's capsule fibroblasts. CONCLUSIONS: The present investigation strongly supports the hypothesis that PDGF-isoforms are major stimulators of proliferation of Tenon's capsule fibroblasts after glaucoma filtering surgery while TGF-beta-isoforms are essential for the transformation of Tenon's capsule fibroblasts into myofibroblasts.