细胞因子及苏拉明对视网膜色素上皮细胞增殖的影响

目的：研究在血小板源性生长因子（PDGF）和白介素-1β（IL-1β）作用下苏拉明（suramin）对培养的人视网膜色素上皮（retinal pigment epithelium,RPE）细胞增殖的影响。方法：将不同浓度的（15,150,250mg／L）苏拉明分别加入用PDGF 10μg／L或IL-1 10μg／L培养的RPE细胞培养液中,继续培养3d后采用四甲基偶氮唑盐（tetrazolium,MTT）比色法,细胞分裂指数计数和核仁组成区嗜银染色（AgNORs）检测在细胞因子作用下不同浓度苏拉明对RPE增殖活力的影响。结果：含有PDGF 10μg／L或IL-1β10μg／L的培养液显著刺激RPE的增殖,苏拉明明显抑制了这两种细胞因子条件下RPE的增殖,在150mg／L时的抑制率分别为26％$H18％,对细胞的形态无明显影响。结论：苏拉明对PDGF或IL-1β刺激的RPE的增殖有显著抑制作用,该作用为非毒性作用。进一步证明苏拉明对增生性玻璃体视网膜病变（proliferative vitreoretinopathy,PVR）中相关细胞因子的拮抗作用,为临床防治PVR提供了新的用药思路。     

苏拉明对体外培养的视网膜色素上皮细胞增殖的影响

目的探讨苏拉明(suram in)对体外培养的猪视网膜色素上皮(retinal p igm ent ep ithelium,RPE)细胞增殖的影响。方法不同浓度苏拉明(0.05、0.1、0.2、0.4g/L)作用于RPE细胞,采用生长曲线法及MTT比色法检测苏拉明对RPE细胞增殖的影响,台盼蓝染色检测细胞活性;流式细胞仪检测细胞周期变化;透射电镜观察各浓度苏拉明作用后细胞超微结构变化。结果苏拉明对RPE细胞增殖有抑制作用(P<0.05),且呈时间剂量效应;流式细胞术检测示苏拉明将细胞阻滞于G2/M期(P<0.01);透射电镜结果示苏拉明浓度为0.05~0.2 g/L时,RPE细胞结构无明显变化,而苏拉明0.4g/L时,细胞表面微绒毛减少,染色质边集。结论苏拉明能够抑制体外培养的猪RPE细胞增殖,但随浓度增加,对细胞有潜在毒性作用。     

苏拉明对体外培养的人视网膜色素上皮细胞增殖的影响

目的 观察苏拉明(suramin)对体外培养的人视网膜色素上皮细胞(retinalpigment epithelial cells)生长、增殖的影响。方法 以300mg·L~(-1)苏拉明作用于培养的视网膜色素上皮细胞,于不同的时间点行细胞计数,绘制生长曲线,台盼蓝染色判定细胞的活性;将不同浓度的苏拉明(0、100、200、300、400mg·L~(-1))加入RPE细胞培养液,72h后用四甲基唑盐(MTT)比色法测吸光值A,并计算不同浓度条件下的细胞增殖抑制率;流式细胞技术检测细胞周期变化。结果 苏拉明在所设浓度内均抑制RPE细胞增殖,并存在时间-效应及剂量-效应关系,最大增殖抑制率约78％,72h时IC_(50)为272mg·L~(-1)。FCM示suramin使G_2/M期细胞增加了14.2％。结论 苏拉明能抑制体外培养的人视网膜色素上皮细胞增殖,可作为防治增生性玻璃体视网膜病变(proliferative vitreoretinopathy,PVR)的候选药物作进一步研究。     

苏拉明联合地塞米松对体外培养的视网膜色素上皮细胞增殖的影响

目的:探讨苏拉明(suramin,Sur)联合地塞米松(dexamethasone,Dex)对体外培养的猪视网膜色素上皮(retinal pigment epithelium,RPE)细胞增殖的影响。方法:选择0.05,0.1,0.2,0.4g/L Sur单独作用及联合0.2g/LDex作用于RPE细胞4d,MTT比色法检测对RPE细胞增殖的影响;0.2g/L Sur和0.2g/L Dex分别作用于RPE细胞,流式细胞术检测细胞周期变化;Sur浓度为0.2g/L,0.4g/L及0.2g/L Sur联合0.2g/L Dex时,透射电镜观察细胞超微结构情况。结果:MTT比色法检测表明各浓度Sur对RPE细胞有抑制作用(P<0.05),呈剂量效应关系;0.2g/L Dex与各浓度Sur联合应用对RPE细胞的抑制率由单独应用Sur的16.0%,27.3%,40.5%,64.1%增至42.3%,52.1%,65.6%,78.8%。流式细胞检测提示Sur将细胞阻滞于G2/M期(P<0.01);Dex将细胞阻滞于S期(P<0.01)。透射电镜结果显示,Sur浓度为0.2g/L时,单独应用及联合0.2g/L Dex应用,RPE细胞结构无明显变化,而Sur0.4g/L时,细胞表面微绒毛减少,细胞核皱缩。结论:Sur联合Dex能有效抑制体外培养的RPE细胞增殖。     

抗CD81抗体对培养大鼠RPE增殖性的影响

目的:观察抗CD81抗体EAT2和H121对培养大鼠视网膜色素上皮细胞(retinalpigmentepithelium,RPE)增殖活性的影响。方法:分别将2,10mg/L抗CD81抗体EAT2与H121加入体外培养大鼠RPE中,7d后以MTT法测定细胞生长抑制率。结果:加入抗CD81抗体后RPE增殖活性下降,10mg/LEAT2对RPE的细胞生长抑制率达72.7%。抑制作用的强弱随抗体种类及抗体浓度的不同而不同。结论:抗CD81抗体EAT2和H121可以抑制培养大鼠RPE的细胞增殖活性。     

姜黄素和苏拉明抑制兔RPE细胞增生的对比研究

目的 对比研究姜黄素和苏拉明对兔视网膜色素上皮(RPE)细胞增生的抑制作用.以苏拉明为对照药,探讨姜黄素防治增生性玻璃体视网膜病变(PVR)的潜在价值.方法 MTT法检测不同质量浓度姜黄素和苏拉明在各时间段对体外培养的RPE细胞增生的抑制率.相关回归分析计算两种药物各时间段的半数抑制率(IC50)剂量.流式细胞仪检测姜黄素和苏拉明对RPE细胞周期及细胞凋亡的影响,并且检测姜黄素作用不同时间后RPE细胞的凋亡率.结果 姜黄素和苏拉明对RPE细胞均有明显的抑制作用,呈时间和质量浓度依赖性.姜黄素在24、48、72及96h的IC50均明显低于苏拉明.姜黄素使RPE细胞阻滞在G0/G1期,早于苏拉明的G2/M期.姜黄素可以诱导RPE细胞凋亡,苏拉明则不能.姜黄素15μg/mL作用24、48及72h后,RPE细胞的凋亡率分别为(12.83±0.13)%、(32.27±4.51)%、(56.81±8.67)%.结论 姜黄素通过诱导RPE细胞凋亡而有效抑制其增生,比苏拉明药效强且起效快,有望成为防治PVR的理想天然药物.     

IL-1ra及地塞米松对IL-1β诱导的人RPE增生的抑制

目的:观察IL-1ra及地塞米松对IL-1β诱导的RPE增生的抑制作用.方法:通过MTT比色实验观察IL-1ra及地塞米松对IL-1β促RPE增生的抑制作用.结果:IL-1ra(20～200 μ g/L)对加入IL-1β(2 μ g/L)培养的人RPE增生有明显的抑制作用,抑制率为9.4%～24.5%(P＜0.05);地塞米松在低浓度(10 mg/L)时刺激RPE增生,而在高浓度(35～70 mg/L)则表现为对IL-1β诱导RPE增生的抑制作用,抑制率为31.2%～43.9%(P＜0.05);IL-1ra(100 μg/L)联合地塞米松(35 mg/L)对IL-1β诱导RPE增生的抑制率40.6%,较两者单独使用的抑制率均高(P＜0.05).结论:IL-1β能促进培养的人RPE的增生,而IL-1ra及地塞米松可以抑制这种促增生作用.     

曲安奈德体外对人视网膜色素上皮细胞功能和结构的影响

目的:探讨曲安奈德(triamcinolone acteonide,TA)对人视网膜色素上皮(retinal pigment epithelium,RPE)细胞功能和活性的影响.方法:通过MTT(四甲基偶氮唑盐比色法),细胞移行实验,细胞黏附实验,RT-PCR检测不同浓度TA(包括含赋形剂的和去除赋形剂的,以及纯赋形剂)在不同时间段对RPE细胞增殖,RPE细胞移行,RPE细胞黏附和线粒体DNA(mitochondrialDNA,mtDNA)损伤的影响.结果:浓度大于0.1g/L含赋形剂和不含赋形剂的TA以及单纯赋形剂对细胞增殖有明显的抑制作用(P＜0.05).细胞移行实验:0.1g/L浓度TA在3个时间点与含血清对照组相比,对100mL/L血清刺激的RPE细胞的移行有显著的抑制作用(P＜0.05).在24,48h与含血清对照组相比各实验组对RPE细胞的黏附有显著的抑制作用(P＜0.05).不同浓度TA(含赋形剂)与RPE细胞作用24h后,结果mtDNA缺失片段不明显,未见明显差异,长片段明显,各组无明显差异.结论:TA对RPE细胞功能(移行能力,黏附能力,增殖能力)有抑制作用,对RPE细胞mtDNA影响不明显.     

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)     

增生细胞核抗原在视网膜色素上皮细胞表达及其反义寡核苷酸的抑制作用

目的 研究视网膜色素上皮(retinal pigment epithelium,RPE)细胞中增生细胞核抗原(proliferating cell nuclear antigen,PCNA)表达及其反义寡核苷酸(antisense oligodeoxynucleotides,AS ODN)对其表达和细胞增生的抑制作用，为增生性玻璃体视网膜病变(proliferative vitreoretinopathy,PVR)治疗探索基因治疗新途径。 方法 (1)体外培养兔眼RPE细胞，在不同时间采用链霉亲合素-生物素化过氧化物酶复合物(streptoavidin-biotin enzyme complex,SABC)免疫组织化学法检测PCNA的表达;(2)脂质体介导下不同浓度的PCNA AS-ODN和正义寡核苷酸(sense oligodeoxynucleotides,S-ODN)分别作用于体外培养的RPE细胞，采用免疫组织化学方法检测PCNA的表达;(3)四唑盐比色法(methyl thiazolyl tetrazolium,MTT)检测在不同浓度的PCNA AS-ODN和S-ODN作用下RPE细胞生长活性及其生长抑制率。 结果 (1)体外培养兔眼RPE细胞表达PCNA，表达高峰为培养后48 h ;(2)在0.28、1.12 μmol/L AS-ODN 作用下，PCNA的表达明显受抑制;(3)0.28、1.12 μmol/L的PCNA AS-ODN 能明显抑制RPE细胞增生活性，并呈剂量依赖性，其生长抑制率分别达53%、81%。 结论 一定浓度PCNA AS-ODN能序列特异性地抑制RPE细胞PCNA表达和增生活性，有望进一步用于PVR的治疗实验研究。 (中华眼底病杂志, 2002, 18: 231-233)     

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)     

Migration of retinal pigment epithelial cells in vitro modulated by monocyte chemotactic protein-1: enhancement and inhibition

BACKGROUND: The migration of retinal pigment epithelial (RPE) cells is an initial step in the development of proliferative vitreoretinopathy (PVR). This in vitro study was carried out to investigate the effects of monocyte chemotactic protein-1 (MCP-1) on the migration and proliferation of RPE cells. METHODS: We used an in vitro wound healing model in which a small area of a confluent monolayer of human RPE (HRPE) cells was denuded with a razor blade. The cultures were subsequently incubated with MCP-1, IL-1beta, TNF-alpha, or combinations thereof. Neutralizing IgG1 of antihuman MCP-1, dexamethasone (DEX) or daunorubicin were also added to the cultures to test their inhibitory effects on migration of RPE cells. HRPE migration was measured as the number of cells that entered the denuded area. The effect of MCP-1 on proliferation of HRPE cells was examined by MTT assay. RESULTS: MCP-1 stimulated HRPE cell migration in a dose-dependent manner. IL-1beta or TNF-alpha slightly stimulated HRPE cell migration, but adding anti-MCP- IgG1 significantly reduced this effect. MCP-1-induced migration could be inhibited by DEX but not by daunorubicin. MCP-1 did not show a significant effect on HRPE cell proliferation. CONCLUSION: MCP-1 stimulates HRPE cell migration, suggesting that this chemokine regulates the development of PVR at the initial stage. The migration of HRPE cells induced by IL-1beta and TNF-alpha may be associated with the MCP-1 that HRPE cells secretes under the stimulation of these two cytokines. The knowledge that MCP-1-induced migration of HRPE cells is inhibited by DEX may be useful in devising an effective treatment for PVR.     

Hepatocyte growth factor stimulates proliferation and migration during wound healing of retinal pigment epithelial cells in vitro

PURPOSE: A defect in retinal pigment epithelial (RPE) cells may cause dysfunction of the neural retina, so rapid recovery of differentiated RPE cells is required after RPE injury. We investigated the effect of hepatocyte growth factor (HGF) on wound healing in RPE cells. METHODS: Confluent monolayers of bovine RPE cells were denuded, and the cells were allowed to recover in the presence or absence of HGF. The effect of HGF on RPE cell proliferation was evaluated by a 3-(4;5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetraz olium assay. In a migration assay, mitomycin C was used to inhibit proliferation, and the number of migrated cells was counted. The signaling pathways involved were examined using inhibitors of mitogen-activated protein kinase (MAPK), phosphatidylinositol-3 (PI3) kinase and protein kinase C pathways. RESULTS: At 80 ng/mL, HGF stimulated the wound closure of RPE monolayers and rendered the restituted cells more epithelioid in shape. HGF at 10 ng/mL stimulated RPE cell migration the most, whereas 80 ng/mL of HGF inhibited migration, but stimulated proliferation the most. In particular, PI3 kinase and MAPK inhibitor inhibited PRE cell migration and proliferation, respectively. CONCLUSIONS: HGF stimulated wound closure in cultured RPE cells, and rendered restituted cells epithelioid in shape. HGF may become a therapeutic candidate for RPE wound healing.     

Epidermal growth factor receptor in cultured human retinal pigment epithelial cells

BACKGROUND: Migration and proliferation of retinal pigment epithelial (RPE) cells play an important role in proliferative vitreoretinopathy. Epidermal growth factor receptor (EGFR) is a cell surface receptor with intrinsic tyrosine kinase activity. The engagement of the receptor by its ligand can induce intracellular mitogenic signal transduction pathways and stimulate proliferation, migration and differentiation of cells. This experiment aimed to investigate the activation and role of EGFR signal transduction pathway in proliferation of human RPE cells. METHODS: Cultured human RPE cells of the 3rd to 6th passages were studied by colorimetric assay for cellular growth and survival (MTT assay) to test the effects of EGF (0.1, 1, 10, 50, and 100 ng/ml) and fetal bovine serum (FBS) on proliferation of human RPE cells. An in vitro wound healing model was also set up, and the number of cells that had entered the denuded area was counted. The human RPE cells were cultured for 3 days with 0.1% FBS, 10% FBS, 10 ng/ml EGF + 0.1% FBS and a combination of EGF and 10% FBS, respectively. Immunohistochemical staining and in situ hybridization were used to observe the expressions of EGFR protein and mRNA, respectively. Activation of mitogen-activated protein kinase (MAPK) was detected by immunohistochemical method with specific antiphosphorylated extracellular signal-regulated kinase (ERK)1/2 antibody. RESULTS: EGF stimulated proliferation and migration of cultured human RPE cells in a concentration-dependent manner. The maximum of the proliferation rate of RPE cells was 81.8% with EGF at a concentration of 10-100 ng/ml of EGF in serum-free Dulbecco's modified essential medium (DMEM) and 122.7% at a concentration of 1-10 ng/ml of EGF in 5% FBS DMEM (p < 0.001); there was a significant difference between serum-free DMEM groups and 5% FBS DMEM groups. The maximum of the migration rate of the cells was 438.9% at a concentration of 10-100 ng/ml of EGF in 10% FBS DMEM, 147% with 10% FBS, and only 36% with EGF in 0.1% FBS at the concentration of 10 ng/ml (p < 0.001). EGF promoted the expression of EGFR protein and mRNA in RPE cells. FBS cooperated with EGF in the stimulation of EGFR expression, and it had a stronger effect in the process than EGF alone. After 3 days of incubation with EGF, phosphorylated ERK1/2 was detectable in the nucleus of RPE cells, whereas cells presented immunostaining positive for phosphorylated ERK1/2 in the cytoplasm before stimulation, indicating that EGF could induce MAPK nuclear translocation. CONCLUSION: EGF could induce EGF-EGFR-MAPK signal transduction pathway in human RPE cells in a concentration-dependent manner in vitro, which may play a key role in the activation of human RPE cell proliferation and migration.     

舒拉明对碱性成纤维细胞生长因子促进人视网膜色素上皮细胞增殖作用拮抗效应的研究

目的探讨舒拉明抗增生性玻璃体视网膜病变(PVR)的作用及机制。方法采用体外培养的人眼视网膜色素上皮(RPE)细胞,总共10组。其中5个对照组的培养基中分别加入终浓度为1、10、100、500ng/ml的bFGF和3125μg/ml的舒拉明。实验组4组,培养基中除加入3125μg/ml的舒拉明外,还分别加入终浓度为1、10、100和500ng/ml的bFGF。另设1组空白组。培养48h后,采用MTT法,酶联免疫检测仪测定各孔吸光度(A)值,计算细胞的增殖率。采用析因分析比较bFGF和舒拉明的交互效应和单独效应。结果各组细胞生长无明显的形态学差异。舒拉明与bFGF存在拮抗性交互效应(F=673,P<001)。对照组比较空白组,全部bFGF组A值都增加,最大A值为bFGF的10ng/ml浓度组;舒拉明组则降低;实验组中,各组A值较bFGF对照组都下降,最大A值左移对应bFGF的100ng/ml浓度组。结论舒拉明能够拮抗bFGF促RPE细胞增殖的作用,其抑制RPE细胞增殖并进一步防治PVR的机制至少包括拮抗了生长因子的生物学效应。     

The effects of anti-vascular endothelial growth factor agents on human retinal pigment epithelial cells under high glucose conditions

AIM: To investigate the effects of high glucose levels and anti-vascular endothelial growth factor (VEGF) agents (bevacizumab, ranibizumab and aflibercept) on retinal pigment epithelium (RPE) cells. METHODS: ARPE-19 cells were cultured at different glucose levels (5.5 mmol/L, 25 mmol/L, and 75 mmol/L). Cell viability was evaluated by MTT assay at 3d after treatment with D-glucose. Cell migration ability was measured by wound healing assay at 3d. A cell death detection kit was used to assess apoptosis at 3 and 14d. Cell proliferation was assessed by EdU assay at 3d. The culture medium was treated with anti-VEGF agents at clinically relevant concentrations. The experiment was then repeated at a different glucose level. RESULTS: The viability and migration of ARPE-19 cells were significantly decreased in the presence of 75 mmol/L as compared to 5.5 mmol/L glucose. The percentage of TUNEL-positive cells was significantly increased and the proliferative potential was decreased with 75 mmol/L compared to 5.5 mmol/L glucose. There were no significant differences in the results between 25 mmol/L and 5.5 mmol/L glucose. In the presence of 75 mmol/L glucose, the groups treated with anti-VEGF showed decreased cell viability and proliferation and increased apoptosis. However, there were no significant differences between the anti-VEGF groups. CONCLUSION: High glucose level decreases the viability, wound healing ability, and proliferation of RPE cells, while increasing apoptosis. Furthermore, anti-VEGF agents interfered with the physiological functions of RPE cells under high-glucose conditions, accompanied by decreases in cell viability and proliferation.     

叶黄素对牛晶状体上皮细胞增殖及迁移的影响

目的观察叶黄素（lutein）对体外培养的牛晶状体上皮细胞（bovine lens epithelial ceHs，BLECs）增殖和移行的影响．为药物防治后发性白内障（after-cataract）提供新的选择。方法首先进行BLECs原代培养和传代培养。取第2～3代BLECs。分别采用MTT法、划线法．研究不同浓度及作用时间的叶黄素对体外培养的BLECs增殖及移行的影响。结果①与对照组相比．当浓度〉μmol／L时，叶黄素能够明显抑制BLECs的增殖．差异有显著性（P〈0．01）。相同作用时间不同浓度组之间抑制作用比较，随药物浓度的增加，抑制作用加强，各浓度组之间差异有显著性（P〈0．01）。而相同药物浓度组随着作用时间的延长，抑制作用加强，各时间点差异亦有显著性（P〈0．01）。②浓度为1μmol和2μmol／L的叶黄素能明显抑制BLECs的的移行，与对照组相比，差异有显著性（P〈0．01）。两组间愈合率比较，差异亦有显著性（P〈0．01）。结论①在浓度≥1μmol时，叶黄素能有效抑制BLECs的增殖。②浓度为1μmol／L和2μmol的叶黄素能抑制BLECs的移行，其强度呈一定时间依赖性。③叶黄素可能成为药物治疗后发性白内障的新选择。