环孢霉素A对体外培养人视网膜色素上皮细胞的抑制

为寻找抑制玻璃体内细胞增生的有效药物,用³H-胸腺嗜啶核苷(²H—TdR)掺入及液体闪烁技术,观察了环孢霉素A(CsA)(0.125"μg/ml~4.0μg/ml)对体外培养的加或不加巨噬细胞培养调理液(MCM)的人视网膜色素上皮(RPE)细胞增生的影响。CsA在0.125μg/ml时细胞的抑制率为-3.8%~4.1%(P>0.05);在0.25μg/ml～4.0μg/ml时为8.4%~95.1%(P<0.05),且呈剂量依赖性(r＝0.9413)(P<0.001),ID₅₀为1.49μg/ml。加MCM组细胞增殖较对照组明显（3d,26%;5d,34%;P<0.05),CsA对细胞增殖抑制率较对照组有提高(1.0%～13.9%)．表明CsA对体外培养的人RPE细胞有明显的抑制作用． （中华眼底病杂志,1995,11：22-24）     

苏拉明对RPE移行和增殖抑制作用的对照研究

目的:进一步研究苏拉明(suramin)对体外培养的人视网膜色素上皮细胞(RPE)移行和增殖的抑制作用。方法:(1)在RPE损伤愈合模型中观察150mg/L suramin对RPE移行的抑制作用。(2)采用四甲基偶氮唑盐比色法(MTT)测定体外培养的人RPE在150mg/L suramin作用下的增殖活性的抑制。结果:(1)移行实验发现:150mg/L的suramin在24h对RPE的移行抑制率为48%;(2)增殖实验发现,150mg/L的suramin在3d时对RPE的增殖抑制率为51%。结论:suramin可以显著抑制细胞的移行和增殖,对细胞移行能力的抑制发生在抑制增殖作用之前,提示苏拉明对RPE的抑制可能是先抑制细胞的移行,尔后抑制细胞的增殖,为苏拉明的临床应用提供进一步相关依据。     

组织型纤溶酶原激活剂对人视网膜色素上皮细胞的抑制

目的：探讨组织型纤溶酶原激活剂（tissue-type plasminogen activator,tPA)对人视网膜色素上皮（retinal pigment epithelial,RPE)细胞的作用。方法：运用活细胞计数法和甲基噻咪基四唑（methyl thiazolyl tetrazolium,MTT)比色实验观察分析tPA对人RPE细胞增殖的抑制作用,应用流式细胞仪（flow eytometry,FCM)分析人RPE细胞周期。结果：0．1－3μg/ml tPA对人RPE细胞有抑增殖作用,其效应与药物浓度和作用时间呈正向依赖关系（P＜0．01）;5μg/ml tpA对人RPE细胞有致死毒性作用。RCM显示tPA作用后S期细胞增加9．8％（P＜0．05）,G2M期减少6．6％（P＜0．05）。结论：tPA在一定时间和剂量范围内对人RPE细胞具有抑增殖作用,且不对细胞产生致死毒性;tPA主要延迟／阻滞人RPE细胞于S期。     

曲安奈德对PVR中增殖细胞抑制效果的观察

目的：探讨不同浓度曲安奈德对体外培养的视网膜前膜细胞、视网膜色素上皮细胞（RPE）及神经胶质细胞的抑制率差异,找出视网膜前膜细胞达最大抑制率时的最低有效药物浓度,用于指导临床外伤性增生性玻璃体视网膜病(PVR)防治。 方法：用四唑盐(MTT)比色法检测不同浓度曲安奈德(TA)对体外培养的视网膜前膜细胞、RPE及神经胶质细胞的作用。 结果：三种细胞对TA的反应不完全相同,其中视网膜前面细胞达最大抑制率时所需要TA的最低有效药物浓度0.8mg/mL,RPE细胞最大抑制率所需TA最低有效药物为0.4mg/mL,神经胶质细胞最大抑制率所需TA最低有效药物为0.6mg/mL;且抑制率与药物作用时间呈正相关性。 结论：TA能够有效抑制体外培养的视网膜前膜细胞、RPE细胞及神经胶质细胞的增生;抑制视网膜前膜细胞增殖所需TA药物浓度明显高于RPE和神经胶质细胞。     

阿霉素、5-Fu、三尖杉酯碱及去炎松对体外培养的人视网膜色素上皮细胞的抑制

应用3H-胸腺嘧啶核苷（3H－TdR）掺入及液体闪烁测量技术，观察不同浓度的阿霉素，5-氟尿嘧啶（5－Fu）、三尖杉酯碱及去炎松对体外培养的人视网膜色素上皮细胞（RPE）增殖的抑制作用。结果；阿霉素在2～32ng／ml时，对RPE细胞抑制率为85～77.2％，ID50为12ng／ml；5－Fu在0.2～3．2μg／ml时，对RPE细胞抑制率为14．5～81．7％，ID50为0．6μg／ml；三尖杉酯碱在2．5～40ng／ml时，抑制率为18．5～94．1％，ID50为4.5ng／ml；去炎松在200～350μg／ml时对RPE细胞抑制率为37．9～53．5％，ID50为340μg／ml。结论：阿霉素、5－Fu、三尖杉酯碱及去炎松能有效地抑制RPE细胞的增殖。     

In vitro safety of intravitreal moxifloxacin for endophthalmitis treatment

PURPOSE: To investigate the safety of moxifloxacin for intravitreal application in a cell culture model. SETTING: Department of Ophthalmology, Ludwig-Maximilians-Universit?t, Munich, Germany. METHODS: Primary human retinal pigment epithelium (RPE) cells, ARPE19 cells, and primary optic nerve head astrocyte (ONHA) cells were treated with concentrations of moxifloxacin ranging from 10 to 750 microg/mL. Possible toxic effects and median inhibitory concentration were evaluated after 24 hours as well as under conditions of oxidative stress. After treating the RPE and ONHA cell lines with tumor necrosis factor-alpha (TNF-alpha; 10 microg/mL), lipopolysaccharides (LPS; 20 microg/mL), and interleukin-6 (IL-6; 20 microg/mL), the effects of moxifloxacin on cellular viability under conditions of inflammation were investigated. Toxicity was evaluated by measuring the inhibition of RPE cell proliferation with the tetrazolium dye-reduction assay (MTT; 3-[4,5-dimethylthiazol- 2-yl]-2,5-diphenyl tetrazolium bromide). Cell viability was quantified by a microscopic live/dead assay. RESULTS: At concentrations higher than 150 microg/mL, moxifloxacin had adverse effects on primary RPE, ARPE19, and ONHA cell proliferation and viability. Lower concentrations did not affect RPE or ONHA cell proliferation and viability when administered for 24 hours. No significant decrease in proliferation and viability was observed after preincubation with TNF-alpha, LPS, and IL-6 for 24 hours and subsequent treatment with moxifloxacin concentrations of 10 to 150 microg/mL for 24 hours. Hydrogen peroxide exposure did not increase cellular toxicity. CONCLUSIONS: No significant toxicity of moxifloxacin was seen on primary RPE cells, ARPE19 cells, or ONHA cells at concentrations up to 150 microg/mL. Intravitreal use of moxifloxacin up to this concentration may be safe and effective for the treatment of endophthalmitis.     

曲安奈德对正常人视网膜色素上皮细胞

目的研究曲安奈德（TA）对视网膜色素上皮（RPE）细胞的毒性作用。方法四甲基偶氮唑盐比色（MTT）法测定不同浓度(0.4、0.2、0.1、0.05、0.025 mg/ml)TA对RPE细胞增生的影响；流式细胞术检测TA［当生长抑制率达到50%时的药物浓度(IC₅₀)］作用72 h后细胞周期的变化；倒置相差显微镜和透射电子显微镜观察细胞形态和超微结构的改变。结果0.4~0.025 mg/ml浓度范围内，与对照组比较，TA作用后RPE细胞生长均有明显抑制 (P＜0.05)，且呈浓度依赖性。TA组S期细胞较对照组减少10.6%（P＜0.05）。光学显微镜下TA组细胞密度稀疏，形态不规则，有较多突起及细胞空泡。电子显微镜下TA组细胞核染色质分布不均，细胞质空化，甚至可见细胞坏死。结论TA可抑制培养人RPE细胞有丝分裂S期，抑制细胞增生；明显破坏细胞结构甚至导致细胞死亡。(中华眼底病杂志,2005,21:233-236)     

Local immunosuppression prolongs survival of RPE xenografts labeled by retroviral gene transfer

PURPOSE: To determine whether local immunosuppression with Cyclosporin A can influence the survival of human fetal retinal pigment epithelium (RPE) xenografts in the rabbit's subretinal space. METHODS: Cultured human fetal RPE cells were transduced with the gene for green fluorescent protein (GFP) using a lentiviral vector. The RPE was transplanted into the subretinal space of rabbits that received intravitreal cyclosporine either by weekly injections (0. 25-0.5 mg) or by slow release (approximately 2 microg/d) from a capsule sutured into the vitreal cavity after prior cryopexy. The transplanted RPE was followed by GFP fluorescence scanning laser ophthalmoscopy and by histology of the transplant site. RESULTS: RPE xenografts in eyes receiving intravitreal cyclosporine survived longer (several months) than they did in control eyes without cyclosporine. Survival was as long with slow release capsules as it was with weekly intravitreal injections at much higher concentrations of cyclosporine. CONCLUSIONS: Local immunosuppression of the eye with cyclosporine prolongs the survival of RPE xenografts in the subretinal space of rabbits, implying that rejection involves activated T lymphocytes. Local immunosuppression with slow release capsules is as effective as weekly injections at much higher concentrations.     

雷帕霉素对人视网膜色素上皮细胞增生和凋亡的影响

背景雷帕霉素（RAPA）是哺乳动物RAPA靶蛋白（mTOR）特异性抑制剂,能有效抑制晶状体上皮细胞（LECs）及多种肿瘤细胞增生并具有诱导肿瘤细胞凋亡的作用,研究其对视网膜色素上皮（RPE）细胞是否具有类似作用对临床上预防和治疗增生性玻璃体视网膜病变（PVR）具有重要意义。目的研究RAPA对体外培养的人RPE细胞增生和凋亡的影响。方法对人RPE细胞（D407细胞株）进行体外传代培养,按照培养基中加入药物的不同将培养的细胞分为9组,空白对照组进行常规培养;二甲基亚砜（DMSO）对照组在培养基中加入质量分数0．1％。DMSO;实验各组将5、10、20、40、80、160、320nmol／LRAPA分别加入培养基中并分别培养12、24、48h。应用噻唑蓝（MTT）法检测各组吸光度（A490）值并计算各组人RPE细胞增生的抑制率,应用Hoechst染色法检测各组人RPE细胞的凋亡率,评价上述各浓度RAPA作用不同时间后对人RPE细胞增生和凋亡的影响。结果不同浓度RAPA组对人RPE细胞的抑制率随浓度的增高而明显升高,差异有统计学意义（F=484．451,P〈0．01）,20～320nmol／LRAPA组在各时间点所测细胞增生抑制率均明显高于DMSO溶剂对照组,差异均有统计学意义（P〈0．01）。RAPA对细胞增生的抑制率随作用时间的延长明显增加,差异有统计学意义（F=232．262,P〈0．01）,各浓度的RAPA作用24h和48h后细胞增生的抑制率均明显高于12h,差异均有统计学意义（P〈0．05）。与空白对照组及溶剂对照组比较,10nmol／LRAPA作用12、24、48h均有诱导细胞凋亡的作用,差异均有统计学意义（P〈0．05）;20～320nmol／LRAPA组细胞凋亡率明显增加,差异有统计学意义（P〈0．叭）。各浓度RAPA组随作用时间的延长人RPE细胞的凋亡率明显增加（F=625．584,P〈0．01）。Hoechst33258染色可见凋亡细胞核碎裂并呈块状致密浓染,染色质固缩。结论RAPA以浓度和时间依赖的方式抑制体外培养的人RPE细胞增生并诱导其凋亡。     

替德肝素抑制冷冻后视网膜色素上皮细胞分泌肝细胞生长因子的研究

目的探讨替德肝素(tedelparin)在抑制冷冻后的人视网膜色素上皮(humanretinapigmentepithelium,hRPE)细胞分泌肝细胞生长因子(humanhepatocytegrowthfactor,HGF)和生长中的作用。方法体外培养的RPE细胞在-80℃下进行冷冻,冷冻时间分为0、15和60s,随后继续体外培养(体外实验组)和注入正常兔眼玻璃体(体内实验组)并在第6天时取出一些兔眼的玻璃体液加入到正常RPE细胞培养液中孵育48h;每实验组再分为两个亚组替德肝素治疗(终浓度25μl/ml)组和未治疗组,在3d、6d时收集细胞培养上清和玻璃体样本,ELISA法测定HGF含量,MTT法测定48h后RPE细胞的增殖状态。结果在体外实验组,比较未冷冻组,冷冻后的RPE细胞随着冷冻时间延长HGF分泌水平增加(F=2736,P<001),替德肝素干预组HGF分泌水平下降(F=17950,P<001)。在体内实验组(兔玻璃体)随着冷冻时间延长HGF浓度较对照组明显增高(F=624,P<001),当玻璃体液(冷冻15s和60s,第6天)加入到正常RPE细胞培养液48h后刺激细胞增殖(P<001),在替德肝素干预组,细胞增殖明显减弱(F=4490,P<005)。结论冷冻可刺激RPE细胞在体外和体内玻璃体环境中HGF的高分泌,且随着冷冻时间增加更为显著。替德肝素可降低冷冻后RPE细胞分泌HGF水平和抑制促生长环境中的RPE细胞生长,具有预防PVR的作用。     

高糖条件下糖康乐对兔视网膜色素上皮细胞超氧化物歧化酶表达的影响

目的:研究海藻萃取物糖康乐在高糖条件下对兔视网膜色素上皮(RPE)细胞超氧化物歧化酶(SOD)表达的影响。方法:应用体外培养的兔RPE细胞,分成空白对照组、高糖组(用25mmol/L)葡萄糖处理、糖康乐组和牛磺酸组4组(n=6),应用高浓度葡萄糖后,糖康乐组加以不同浓度的糖康乐,同时应用牛磺酸作为阳性对照,测定不同浓度的糖康乐对RPE细胞SOD表达的影响。结果:在高糖条件下,高糖组RPE细胞SOD含量明显降低(2024±91→747±353kat/L)(t=8.570,P<0.001),糖康乐在浓度为0.002mg/L(t=3.207,P<0.01)、0.02mg/L(t=4.235,P<0.01)和0.2mg/L(t=3.748,P<0.01)的时候可以显著地抑制SOD表达的降低,与高糖组相比较有显著的差异,与阳性对照组的牛磺酸组相比,未见明显差异。结论:糖康乐可保护高糖导致的RPE细胞SOD表达的降低。     

肝细胞生长因子对培养的视网膜色素上皮 细胞移行及增生的作用

目的研究肝细胞生长因子(hepatocyte growth factor, HGF)对视网膜色素上皮 (retinal pigment epitheliaum, RPE) 细胞的促增生和促移行作用。方法在无血清培养液培养的人RPE细胞中分别加入1、2、10、50、100 μg/L不同浓度的HGF,四唑盐比色法(methyl thiazolyl tetrazolium, MTT)测定促细胞增生情况; 采用RPE细胞损伤愈合模型,在无血清培养液中分别加入1、2、10、50、100 μg/L 不同浓度的HGF,培养20 h后计数进入刮痕区的细胞,观察HGF对RPE细胞移行的作用。结果HGF浓度处于10 ～100 μg/L时对RPE细胞具有促增生作用(P<0.05或P<0.01), 增生率为18.2 %～34.8 %,其中浓度为50 μg/L作 用3 d达到最大促增生效果(P<0.01）,增生率为32.8 %;HGF可明显促进RPE细胞移行,促移行能力分别为113.0 %(10 μg/L), 91.7 %(50 μg/L) 和50.3 %(100 μg/ L )。浓度自2 μg/L开始出现促细胞移行作用(P<0.05),促移行能力为9.3 %。结论HGF可促进RPE细胞增生和移行,是RPE细胞的有丝分裂原和强力的促移行因子。(中华眼底病杂志,2001,17:307-310)     

透明质酸刺激活性物对人视网膜色素上皮细胞的抑制

目的观察透明质酸(hyaluronic acid,HA)刺激活性物(HA-stimulating activity,HASA)对培养的人视网膜色素上皮(retinal pigment epithelial,RPE)细胞的作用。方法将不同质量浓度的HASA加入RPE细胞培养液, 采用活细胞计数、四甲基偶氮唑盐(tetrazolium,MTT)比色法及氚标胸腺嘧啶核苷(³ H-th ymidine,^3 H-TdR)掺入法检测细胞活力,用流式细胞仪(flow cytometry,FCM)分析RPE细胞周期。结果HASA在12.5～200．0 μg/ml范围及48小时内对人RPE细胞的抑制存在剂量-效应及时间-效应关系。最大细胞抑制率约48.0%。FCM显示 HASA作用后G₁期细胞数较对照组增加7.2%,S期减少9.7%。结论HASA在一定时间和剂量范围内对人RPE细胞有抑制作用。(中华眼底病杂志,1999,15:72-74)     

Role of inhibitors of isoprenylation in proliferation, phenotype and apoptosis of human retinal pigment epithelium

BACKGROUND: Isoprenoid biosynthesis is known to be essential for diverse cellular functions, including cell proliferation. The aim of this work was to study the effects caused by the addition of different inhibitors of isoprenylation (lovastatin, manumycin A, farnesyltransferase inhibitor III and N-acetyl-S-farnesyl-L-cysteine) to human retinal pigment epithelium (RPE) in culture, as potential coadjunctive-to-surgery treatments applicable to proliferative vitreoretinopathy. METHODS: Human RPE cell cultures were established from adult corneal donors. Proliferation levels were evaluated using the incorporation of 5-bromo-2'-deoxyuridine into the DNA. Cell viability was measured by tetrazolium bromide transformation. Apoptosis was determined by DNA fragmentation assay, TdT-mediated d-UTP-X nick-end labeling (TUNEL) and phosphatidylserine exposure assessment. Changes in cell morphology and actin cytoskeleton were evaluated using a phase-contrast microscope and by fluorescent staining of actin cables with TRITC-phalloidin. RESULTS: We found that lovastatin showed an important antiproliferative effect on human RPE cells in culture. This effect was clearly dose-dependent, and adding mevalonate could reverse it. We also found that lovastatin induced changes in the distribution of actin cytoskeleton and, finally, that it also induced RPE apoptosis. Manumycin A, farnesyltransferase inhibitor III and N-acetyl-S-farnesyl-L-cysteine also showed antiproliferative effects in RPE. However, they do not have any effect on cell morphology or induction of apoptosis. DISCUSSION: We identified various effects of lovastatin on human RPE cultures: inhibition of cell proliferation, modifications of the phenotype and induction of apoptosis. Interestingly, the addition of different inhibitors of protein isoprenylation only affected the proliferation of the cells. There was no evidence that isoprenylated proteins inhibition is related to lovastatin-induced RPE apoptosis.     

The effect of short-term exposure of triamcinolone acetonide on fibroblasts and retinal pigment epithelial cells

PURPOSE: We investigated the effects of short-term exposure to triamcinolone on cultured choroidal fibroblast (CFB) cells and retinal pigment epithelial (RPE) cells. METHODS: To evaluate the effect of triamcinolone on cell proliferation, CFB and RPE cells were divided into three groups: a short-term exposure group; a longterm exposure group, and a non-treated control group. Cells in the short-term exposure group were briefly exposed (5, 15 or 30 mins) to triamcinolone (0.01 mg/ml, 1 mg/ml or mitomycin C (0.01 microg/ml, 1 microg/ml). Cells in the longterm exposure group were continuously incubated in culture medium containing the drug until assessment. The control group was cultured without drugs. Cell viability and the number of cells were assessed at day 5 after exposure. To investigate the direct toxicity of triamcinolone on confluent RPE cells, completely confluent cells were exposed to the drugs in the manner as described above. Cell viability was determined on days 0, 3 and 5 after treatment. RESULTS: In the short-term exposure group, 1 mg/ml triamcinolone caused a significant reduction in the proliferation of CFB and RPE cells. The proliferation of CFBs decreased even with exposure to 0.01 mg/ml triamcinolone. In the longterm exposure group, triamcinolone and mitomycin C reduced the proliferation of both CFB and RPE cells. Even very short periods of exposure to triamcinolone caused a significant reduction in the viability of completely confluent RPE cells. CONCLUSIONS: Even short periods of exposure to triamcinolone inhibited the proliferation of fibroblasts and RPE cells and were significantly toxic to completely confluent RPE cells.     

MicroRNA-96对人视网膜色素上皮细胞增殖与迁移的影响

目的：探讨MicroRNA-96（miR-96）对人视网膜色素上皮（RPE）细胞增殖和迁移的影响。方法：实验研究。通过RNA原位杂交检测人胚胎眼（20周）石蜡切片中RPE层miR-96的表达情况。将miR-96和阴性对照（NC）通过阳离子脂质体介导转染人眼RPE细胞,采用细胞增殖实验（MTS）、流式细胞术和Transwell实验分别检测细胞增殖、细胞周期以及细胞迁移能力。通过生物信息学及Western blot法确定miR-96作用的靶基因。应用Western blot检测miR-96对细胞增殖迁移相关信号通路蛋白（Akt、ERK）及细胞周期相关蛋白（p-Cdc2、CyclinD2、p-Rb）表达的影响。组间数据比较采用独立样本t检 验。结果：MiR-96在人眼RPE细胞中有表达。MTS结果显示,转染NC、miR-96后,人眼RPE细胞的相对增殖速率分别为100%、74%±2%,差异有统计学意义（t=42.174,P=0.002）。流式细胞术检测结果显示,转染miR-96后阻滞在G1期的RPE细胞显著多于转染NC后,且差异有统计学意义（t= -18.444,P=0.003）。Transwell实验结果显示,与转染NC相比,转染miR-96能显著抑制RPE细胞的迁移,差异具有统计学意义（t=6.754,P=0.002）。进而,明确了MITF是miR-96作用的靶基因。Western blot检测结果显示,转染miR-96后细胞中细胞周期相关蛋白p-Rb（t=11.211,P=0.002）、p-Cdc2（t=9.133, P=0.003）、CyclinD2（t=7.542,P=0.005）以及迁移相关信号通路蛋白p-ERK（t=16.699,P<0.001）,p-Akt （t=23.552,P<0.001）的表达水平均降低。结论：miR-96通过作用于靶基因MITF,并调控细胞周期和迁移相关蛋白的表达从而抑制人RPE细胞的增殖和迁移。     

Bevacizumab wirkt nicht toxisch auf Zellen des menschlichen Auge

OBJECTIVE: Intravitreal anti-vascular endothelial growth factor (VEGF) treatment with bevacizumab (Avastin) has emerged as a promising therapy for the treatment of choroidal neovascularisation in age-related macular degeneration. Intravitreal administration of bevacizumab is "off-label," and only very limited data regarding short-term toxicity exist. Therefore, we investigated the safety of different doses of bevacizumab on the anterior- and posterior-segment cells of the human eye. METHODS: Primary human retinal pigment epithelium (RPE) cells, human optic nerve head astrocytes (ONHA), human trabecular meshwork cells (TMC), and cornea buttons not suitable for transplantation were treated with bevacizumab (25 microg/ml, 250 microg/ml, and 2,500 microg/ml) for 48 h, corresponding to 0.1x, 1x, and 10x the dosage used intravitreally. Bevacizumab-related toxicity was evaluated by a colorimetric test (MTT) measuring inhibition of RPE, ONHA, and TMC cell proliferation. Additionally, cell viability was quantified by live/dead fluorescence assay. Corneal endothelium was quantified by phase-contrast microscopy. RESULTS: Bevacizumab showed adverse effects on primary RPE cell proliferation as well as on cell viability at a concentration of 2,500 microg/ml. The lower concentrations of 25 microg/ml and 250 microg/ml had no influence on RPE cell proliferation or cell viability. There was no toxicity for any investigated concentration on human ONHA, TMC, or corneal endothelium. CONCLUSION: In this study, a 10-fold concentration (compared with common clinical use) of the VEGF-blocking antibody bevacizumab had toxic effects on primary RPE. There was no toxicity for lower concentrations or toxicity to other cell types of the anterior and posterior segments. Therefore, the clinical use of bevacizumab at concentrations of 1-1.25 mg intravitreally seems to be safe.     

苏拉明对体外培养的人眼视网膜色素上皮 细胞增生抑制作用的时效性研究

目的观察苏拉明对体外培养的人眼视网膜色素上皮（RPE）细胞增生抑制作用的时效关系，了解它对RPE细胞的作用方式，进一步阐明其防治增生性玻璃体视网膜病变（PVR）的优势。方法9块96孔细胞培养板，每块取12孔，其中实验组和对照组各6孔。2组各孔内均接种浓度为5×104个/ml的RPE细胞0.1 ml。换液后实验组加250 μg/ml苏拉明，对照组不加。4 d后2组均更换成正常培养液；在生物倒置显微镜下观察RPE细胞生长情况；分别于加药后1、2、4 d及撤药后1、2、3、5、7、9 d随机抽取1块培养板终止培养，采用四甲基偶氮唑盐（MTT）比色法检测同一培养板上两组RPE细胞增生情况。随机化区组 t 检验比较两组吸光度值的差异，计算细胞增生抑制率。结果倒置显微镜下，对照组RPE细胞在接种后第7天完全融合。实验组细胞间隙较对照组稍增大，接种13 d时细胞仍未融合。实验组RPE细胞加入苏拉明后第1天，增生抑制率为14.85%，第4天达最高25.79%；撤药后第1天下降到12.35%，然后逐渐又上升，到撤药后第3~5天达顶峰超过20%，之后逐渐回落，到第9天达14.71%。结论苏拉明对血清诱导的RPE细胞增生具有长时间的抑制作用，特别是在撤除药物后还能再次引起后抑制作用，整个过程呈现特殊的双峰型抑制效应。(中华眼底病杂志,2005,21:25-27)     

培养人视网膜色素上皮细胞中β-半乳糖苷酶活性研究

目的　观察培养人视网膜色素上皮 (RPE)细胞中复制衰老相关的 β 半乳糖苷酶活性。 方法　在培养的RPE细胞中 ,以传代次数 ( 5、10、2 0 )和取材供体的年龄 ( 2 6、44、5 7岁 )分组 ,进行标准化的 β 半乳糖苷酶活性和细胞增殖能力检测。结果　培养RPE细胞中 β 半乳糖苷酶活性阳性细胞数量随传代次数的增加而增加 ,在 2 0代的细胞中 ,阳性细胞的数量为 87%± 1 2 % ,明显高于第 5代和第 10代细胞 (P <0 0 5 )。而细胞的增殖能力明显下降 (P <0 0 5 )。结论　培养RPE细胞复制衰老的发生与细胞传代次数和取材年龄相关 ,其相关研究可能对了解年龄相关性视网膜疾病的发病机制有帮助     

缺氧对培养人视网膜色素上皮细胞线粒体酶活性的影响

目的　研究缺氧对培养的人视网膜色素上皮(retinalpigmentepithelium ,RPE)细胞线粒体琥珀酸脱氢酶(succinatedehydrogenase,SDH)和细胞色素氧化酶(cytochromeoxi dase,COX)活性的影响,以及缺氧对RPE细胞增生的作用。方法　氯化钴(CoCl2 )法建立培养人RPE细胞缺氧模型。于缺氧后0 .5h、1h、2h、4h、6h、8h、16h和2 4h ,用组织化学法测定SDH和COX活性。于缺氧后第1天、2天、3天、4天和5天,用四甲基偶氮唑蓝[3(4,5dimethylthiazole 2 yl) 2 ,5diphenyltetrazoliumbromide,MTT]比色法检测RPE细胞增生。结果　RPE细胞SDH和COX活性随缺氧时间延长而逐渐降低,与对照组有显著性差异(P <0 .0 5 )。缺氧组RPE细胞增生能力增强(P <0 .0 5 )。结论　缺氧可使RPE细胞线粒体酶活性降低,并刺激RPE细胞增生,对深入了解视网膜脉络膜缺血缺氧性疾病的发病机制有一定的价值