促红细胞生成素对人视网膜色素上皮细胞氧化损伤保护作用及其机制

目的 观察促红细胞生成素（EPO）对人视网膜色素上皮（hRPE）细胞抗过氧化氢（H₂O₂）损伤的影响。方法以传代培养hRPE细胞为研究对象, 采用800μmol/L H₂O₂ 作用3 h 建立 hRPE 细胞损伤模型, 分为正常对照组、单纯损伤组、重组人EPO（rhEPO）治疗组。治疗组又根据加入rhEPO浓度不同分为10、20、40、60 IU/ml亚组。免疫组织化学方法检测细胞中核因子kappa B(NF-κB)的活化情况，比色法测定细胞脂质过氧化产物丙二醛（MDA）含量及乳酸脱氢酶（LDH）细胞释放率。结果 H₂O₂ 可使培养液中MDA及LDH释放率上升，单纯损伤组与正常对照组相比，差异有统计学意义（t_LDH＝29.746，t_MDA＝20.426，P＜0.05）；各治疗组与单纯损伤组比较，MDA及LDH释放率均有显著降低，差异有统计学意义（t_10IU＝5.770，t_20IU＝12.774，t_40IU＝19.818，t_60IU＝24.833，P＜0.05；MDA t_10IU＝5.345，t_20IU＝10.278，t_40IU＝18.571，t_60IU＝20.247，P＜0.05）；各治疗组相关分析显示，rhEPO浓度与LDH细胞释放率及MDA含量呈负相关（r=-0.976，P=0.024； r=-0.968，P=0.032）；rhEPO浓度与NF-κB核移位率呈正相关（r=0.998，P=0.002）；NF-κB核移位率与MDA含量呈负相关（r=-0.954，P=0.046）。结论 EPO能有效地拮抗 H₂O₂ 对hRPE细胞的脂质过氧化损害，其机制可能与活化NF-κB有关。     

促红细胞生成素对人视网膜色素上皮细胞光化学损伤的保护作用

目的 观察重组人促红细胞生成素（EPO）在人视网膜色素上皮(RPE)细胞光化学损伤中的保护作用并探讨其作用机制。 方法 取成人RPE（ARPE）19细胞株传代培养的2～5代细胞建立光损伤模型，光照12 h后再培养24 h终止培养，采用四甲基偶氮唑蓝比色法（MTT）法和流式细胞双染色法检测不同浓度的EPO干预治疗前后RPE细胞活性的变化及细胞凋亡的变化，并添加酪氨酸激酶/信号转导和转录活化因子Jak/STAT的阻断剂（AG490）探讨人重组EPO对人RPE细胞光化学损伤的保护性作用及其保护性机制。 结果 EPO可明显增强 RPE细胞的细胞活性，各组中以40 IU/ml EPO组细胞活性的增强结果最明显；明显减少光化学损伤诱导的人RPE细胞的凋亡，以40 IU/ml EPO组结果最明显。在加入AG490（Jak2激酶抑制剂）组，EPO对细胞活性的增强作用和抑制凋亡作用均被阻止。 结论 EPO对人RPE细胞的光化学损伤有保护治疗作用，其保护作用机制主要通过EPO与其受体结合后保护作用机制起作用。     

重组人促红细胞生成素对人视网膜色素上皮细胞光化学损伤保护性作用的研究

目的研究重组人促红细胞生成素（rhEPO）在人视网膜色素上皮（RPE）细胞光化学损伤中的保护作用及其作用机制,为年龄相关性黄斑变性等眼部病变的药物治疗和预防提供理论依据。方法为实验研究。取成人ARPE-19细胞株传代培养的2～5代细胞建立光损伤模型,光照12h后再培养24h终止培养,采用噻唑蓝比色法和AnnexinV-FITC／PI流式双染法检测不同浓度的rhEPO干预治疗前后RPE细胞活性的变化及细胞凋亡的变化,采用酶联免疫吸附实验和免疫组织化学法定量检测不同含量rhEPO干预治疗前后RPE细胞caspase-3表达的变化,并添加AG490（Jak2激酶抑制剂）探讨重组人EPO对人RPE细胞光化学损伤的保护性作用和途径及其保护性机制。结果rhEPO可明显增强RPE细胞的活性,以40U／mLEPO组结果最明显;明显减少光化学损伤诱导的人RPE细胞的凋亡,以40U／mLrhEPO组结果最明显。在加入AG490组,rhEPO对细胞活性的增强作用和抑制凋亡作用均被阻止。结论rhEPO对人RPE细胞的光化学损伤有保护治疗作用,主要通过EPO的受体后保护作用机制起作用,即通过与受体结合,激活Jak2激酶的途径实现的。（中华眼科杂志,2008,44：50-55）     

雌激素对人视网膜色素上皮细胞氧化损伤的保护作用

目的 探讨雌激素对过氧化氢(H2O2)氧化损伤人视网膜色素上皮(RPE)细胞的保护作用及其机制. 方法 制作人RPE细胞氧化损伤模型,分为对照组、H2O2损伤组和预处理组,后者于损伤前24 h加入17β-E2,根据17β-E2的浓度分为10-6、10-5、10-4 μmol/L亚组.采用分光光度计测定乳酸脱氢酶(LDH)释放率及细胞脂质过氧化产物丙二醛(MDA)的浓度,细胞免疫组织化学法检测bcl-2的表达情况. 结果 H2O2损伤组与对照组相比,LDH释放率、MDA浓度明显升高,而bcl-2的表达减少,差异有统计学意义(P＜0.05);各预处理组与H2O2损伤组比较,LDH释放率、MDA浓度均有降低,而bcl-2的表达增加,差异有统计学意义(P＜0.05).随17β-E2浓度的增加LDH释放率、MDA浓度减少,bcl-2的表达增加. 结论 雌激素对H2O2诱导的人RPE细胞的氧化损伤有保护作用.     

虾青素对人视网膜色素上皮细胞氧化损伤的保护作用

目的：：探讨虾青素(astaxanthin,AST)对过氧化氢(hydrogen peroxide,H2 O2)诱导人视网膜色素上皮细胞( retinal pigment epithelial cells,RPE)氧化损伤的保护作用。方法：人RPE细胞系传代培养,MTT检测细胞活力,流式细胞仪检测细胞凋亡率,透射电镜观察超微结构变化。结果：MTT结果显示用10-8 mol/L和10-4 mol/L AST处理后, RPE 细胞活性分别提高到53.66％±3.25％和70.43％±2.38％,与氧化损伤组(38.76％±3.74％)比较,差异具有统计学意义(P<0.05);流式细胞计数结果显示,预先给予10-8 mol/L和10-4 mol/L的AST作用后, RPE细胞凋亡率分别下降到30.23％±1.91％和12.58％±2.12％,与氧化损伤组(42.50％±1.94％)比较,差异具有统计学意义( P<0.05)。电镜观察结果显示,伴随AST作用浓度的增加,细胞形态亦逐渐得到改善。结论：AST可以抑制H2 O2诱导的人RPE细胞的凋亡,从而为寻求有效的防治视网膜损伤的药物提供可靠的实验依据。     

过氧化氢诱导人视网膜色素上皮细胞转录和表达促红细胞生成素的研究

目的研究不同浓度的过氧化氢诱导人视网膜色素上皮（RPE）细胞转录和表达促红细胞生成素（EPO）的现象并探讨其发生机制。方法原代培养人胚胎RPE细胞,经鉴定后,分别应用0、200、400、600、800μmol／L浓度的过氧化氢作用6h,造成RPE细胞不同程度的氧化应激,测定细胞内超氧化物歧化酶（SOD）和丙二醛（MDA）含量;采用半定量RT-PCR法检测RPE细胞氧化应激中EPO在转录水平的表达变化;免疫组织化学法检测EPO在RPE细胞中的表达部位和表达变化。结果过氧化氢导致RPE细胞内SOD含量下降,MDA含量升高。半定量RT-PCR法检测结果显示过氧化氢诱导EPOmRNA表达量增加,并在一定范围内随着过氧化氢浓度的增高而增加,在600μmol／L过氧化氢组EPOmRNA表达量达最高值,之后随着浓度的上升而下降。免疫组织化学染色法检测结果显示过氧化氢可以诱导人RPE细胞EPO表达增强,并在一定范围内随着过氧化氢浓度的增高而增强,在600μmol／L过氧化氢组EPO表达量达到高峰。结论过氧化氢导致的氧化应激可以诱导人RPE细胞转录和表达EPO,EPO的表达量与RPE存活和耐受程度有关。     

黄酮对视网膜色素上皮细胞氧化损伤的保护作用

目的:观察黄酮对氧化剂所诱导的视网膜色素上皮细胞(RPE)的保护作用。方法:在体研究中,预先给予5g/L黄酮滴眼液(3次/d),1wk后舌下静脉注射NaIO3诱导大鼠RPE变性,在2和4wk末,采用视网膜电图(ERG)测量C波。离体研究中,采用缺氧、H2O2、NaN3和t-BHP诱导RPE细胞损伤,并用MTT法检测细胞的存活率。结果:ERG的C波结果表明,第4wk末,黄酮抑制了由NaIO3诱导的大鼠RPE变性。离体研究结果表明,黄酮对多种氧化剂所诱导的RPE细胞损伤具有保护作用。结论:黄酮对氧化诱导的在体和离体视网膜色素上皮细胞均具有保护作用。     

褪黑素对体外培养的人视网膜色素上皮细胞氧化损伤的保护作用

Objective To investigate the protective effects of melatonin on the retinal pigment epithelium (RPE) against oxidative damage induced by hydrogen dioxide (H2O2) and its mechanism. Methods The RPE cells were seeded and divided into normol control group, oxidative damage group and the treatment group (treated with melatonin at the concentration of 1×10.7 mol/L,1×10-6 mol/L, 1×10-5 mol/L and 1×10-4 mol/L). The model of oxidative damage on the RPE cells was established by culturing the RPE cells with H2O2 at the concentration of 600 μmol/L for 1 hour in vitro. The cell viability of RPE cells was detected by the methyl thiazolyl tetrazolium (MTT) method. The degree of oxidative damage was evaluated by detecting the superoxide dismutase (SOD) and maleic dialdehyde (MDA). Apoptosis was detected qualitatively using the DNA Ladders electrophoretic mothed, and quantitatively using the Annexin V-FITC/PI double staining flow cytometry. Results Compared with normal control group, the oxidative damage group had low cell viability, low SOD and high MDA contents, and high apoptosis rate(t=2.25,39.50,68.42;P<0.05). Compared with oxidative damage group, the treatment group had high cell viability, high SOD and low M DA contents, and low apoptosis rate (P<0.05). Conclusions Melatonin has a protective effect on the RPE against oxidative damage induced by H2O2. The mechanism may involve in reinforcing the cell viability, strengthening the activity of antioxidase, and reducing the apoptosis.     

褪黑素对体外培养的人视网膜色素上皮细胞氧化损伤的保护作用

Objective To investigate the protective effects of melatonin on the retinal pigment epithelium (RPE) against oxidative damage induced by hydrogen dioxide (H2O2) and its mechanism. Methods The RPE cells were seeded and divided into normol control group, oxidative damage group and the treatment group (treated with melatonin at the concentration of 1×10.7 mol/L,1×10-6 mol/L, 1×10-5 mol/L and 1×10-4 mol/L). The model of oxidative damage on the RPE cells was established by culturing the RPE cells with H2O2 at the concentration of 600 μmol/L for 1 hour in vitro. The cell viability of RPE cells was detected by the methyl thiazolyl tetrazolium (MTT) method. The degree of oxidative damage was evaluated by detecting the superoxide dismutase (SOD) and maleic dialdehyde (MDA). Apoptosis was detected qualitatively using the DNA Ladders electrophoretic mothed, and quantitatively using the Annexin V-FITC/PI double staining flow cytometry. Results Compared with normal control group, the oxidative damage group had low cell viability, low SOD and high MDA contents, and high apoptosis rate(t=2.25,39.50,68.42;P<0.05). Compared with oxidative damage group, the treatment group had high cell viability, high SOD and low M DA contents, and low apoptosis rate (P<0.05). Conclusions Melatonin has a protective effect on the RPE against oxidative damage induced by H2O2. The mechanism may involve in reinforcing the cell viability, strengthening the activity of antioxidase, and reducing the apoptosis.     

褪黑素对体外培养的人视网膜色素上皮细胞氧化损伤的保护作用

Objective To investigate the protective effects of melatonin on the retinal pigment epithelium (RPE) against oxidative damage induced by hydrogen dioxide (H2O2) and its mechanism. Methods The RPE cells were seeded and divided into normol control group, oxidative damage group and the treatment group (treated with melatonin at the concentration of 1×10.7 mol/L,1×10-6 mol/L, 1×10-5 mol/L and 1×10-4 mol/L). The model of oxidative damage on the RPE cells was established by culturing the RPE cells with H2O2 at the concentration of 600 μmol/L for 1 hour in vitro. The cell viability of RPE cells was detected by the methyl thiazolyl tetrazolium (MTT) method. The degree of oxidative damage was evaluated by detecting the superoxide dismutase (SOD) and maleic dialdehyde (MDA). Apoptosis was detected qualitatively using the DNA Ladders electrophoretic mothed, and quantitatively using the Annexin V-FITC/PI double staining flow cytometry. Results Compared with normal control group, the oxidative damage group had low cell viability, low SOD and high MDA contents, and high apoptosis rate(t=2.25,39.50,68.42;P<0.05). Compared with oxidative damage group, the treatment group had high cell viability, high SOD and low M DA contents, and low apoptosis rate (P<0.05). Conclusions Melatonin has a protective effect on the RPE against oxidative damage induced by H2O2. The mechanism may involve in reinforcing the cell viability, strengthening the activity of antioxidase, and reducing the apoptosis.     

褪黑素对体外培养的人视网膜色素上皮细胞氧化损伤的保护作用

Objective To investigate the protective effects of melatonin on the retinal pigment epithelium (RPE) against oxidative damage induced by hydrogen dioxide (H2O2) and its mechanism. Methods The RPE cells were seeded and divided into normol control group, oxidative damage group and the treatment group (treated with melatonin at the concentration of 1×10.7 mol/L,1×10-6 mol/L, 1×10-5 mol/L and 1×10-4 mol/L). The model of oxidative damage on the RPE cells was established by culturing the RPE cells with H2O2 at the concentration of 600 μmol/L for 1 hour in vitro. The cell viability of RPE cells was detected by the methyl thiazolyl tetrazolium (MTT) method. The degree of oxidative damage was evaluated by detecting the superoxide dismutase (SOD) and maleic dialdehyde (MDA). Apoptosis was detected qualitatively using the DNA Ladders electrophoretic mothed, and quantitatively using the Annexin V-FITC/PI double staining flow cytometry. Results Compared with normal control group, the oxidative damage group had low cell viability, low SOD and high MDA contents, and high apoptosis rate(t=2.25,39.50,68.42;P<0.05). Compared with oxidative damage group, the treatment group had high cell viability, high SOD and low M DA contents, and low apoptosis rate (P<0.05). Conclusions Melatonin has a protective effect on the RPE against oxidative damage induced by H2O2. The mechanism may involve in reinforcing the cell viability, strengthening the activity of antioxidase, and reducing the apoptosis.     

褪黑素对体外培养的人视网膜色素上皮细胞氧化损伤的保护作用

Objective To investigate the protective effects of melatonin on the retinal pigment epithelium (RPE) against oxidative damage induced by hydrogen dioxide (H2O2) and its mechanism. Methods The RPE cells were seeded and divided into normol control group, oxidative damage group and the treatment group (treated with melatonin at the concentration of 1×10.7 mol/L,1×10-6 mol/L, 1×10-5 mol/L and 1×10-4 mol/L). The model of oxidative damage on the RPE cells was established by culturing the RPE cells with H2O2 at the concentration of 600 μmol/L for 1 hour in vitro. The cell viability of RPE cells was detected by the methyl thiazolyl tetrazolium (MTT) method. The degree of oxidative damage was evaluated by detecting the superoxide dismutase (SOD) and maleic dialdehyde (MDA). Apoptosis was detected qualitatively using the DNA Ladders electrophoretic mothed, and quantitatively using the Annexin V-FITC/PI double staining flow cytometry. Results Compared with normal control group, the oxidative damage group had low cell viability, low SOD and high MDA contents, and high apoptosis rate(t=2.25,39.50,68.42;P<0.05). Compared with oxidative damage group, the treatment group had high cell viability, high SOD and low M DA contents, and low apoptosis rate (P<0.05). Conclusions Melatonin has a protective effect on the RPE against oxidative damage induced by H2O2. The mechanism may involve in reinforcing the cell viability, strengthening the activity of antioxidase, and reducing the apoptosis.     

褪黑素对体外培养的人视网膜色素上皮细胞氧化损伤的保护作用

Objective To investigate the protective effects of melatonin on the retinal pigment epithelium (RPE) against oxidative damage induced by hydrogen dioxide (H2O2) and its mechanism. Methods The RPE cells were seeded and divided into normol control group, oxidative damage group and the treatment group (treated with melatonin at the concentration of 1×10.7 mol/L,1×10-6 mol/L, 1×10-5 mol/L and 1×10-4 mol/L). The model of oxidative damage on the RPE cells was established by culturing the RPE cells with H2O2 at the concentration of 600 μmol/L for 1 hour in vitro. The cell viability of RPE cells was detected by the methyl thiazolyl tetrazolium (MTT) method. The degree of oxidative damage was evaluated by detecting the superoxide dismutase (SOD) and maleic dialdehyde (MDA). Apoptosis was detected qualitatively using the DNA Ladders electrophoretic mothed, and quantitatively using the Annexin V-FITC/PI double staining flow cytometry. Results Compared with normal control group, the oxidative damage group had low cell viability, low SOD and high MDA contents, and high apoptosis rate(t=2.25,39.50,68.42;P<0.05). Compared with oxidative damage group, the treatment group had high cell viability, high SOD and low M DA contents, and low apoptosis rate (P<0.05). Conclusions Melatonin has a protective effect on the RPE against oxidative damage induced by H2O2. The mechanism may involve in reinforcing the cell viability, strengthening the activity of antioxidase, and reducing the apoptosis.     

褪黑素对体外培养的人视网膜色素上皮细胞氧化损伤的保护作用

Objective To investigate the protective effects of melatonin on the retinal pigment epithelium (RPE) against oxidative damage induced by hydrogen dioxide (H2O2) and its mechanism. Methods The RPE cells were seeded and divided into normol control group, oxidative damage group and the treatment group (treated with melatonin at the concentration of 1×10.7 mol/L,1×10-6 mol/L, 1×10-5 mol/L and 1×10-4 mol/L). The model of oxidative damage on the RPE cells was established by culturing the RPE cells with H2O2 at the concentration of 600 μmol/L for 1 hour in vitro. The cell viability of RPE cells was detected by the methyl thiazolyl tetrazolium (MTT) method. The degree of oxidative damage was evaluated by detecting the superoxide dismutase (SOD) and maleic dialdehyde (MDA). Apoptosis was detected qualitatively using the DNA Ladders electrophoretic mothed, and quantitatively using the Annexin V-FITC/PI double staining flow cytometry. Results Compared with normal control group, the oxidative damage group had low cell viability, low SOD and high MDA contents, and high apoptosis rate(t=2.25,39.50,68.42;P<0.05). Compared with oxidative damage group, the treatment group had high cell viability, high SOD and low M DA contents, and low apoptosis rate (P<0.05). Conclusions Melatonin has a protective effect on the RPE against oxidative damage induced by H2O2. The mechanism may involve in reinforcing the cell viability, strengthening the activity of antioxidase, and reducing the apoptosis.     

褪黑素对体外培养的人视网膜色素上皮细胞氧化损伤的保护作用

目的 探讨褪黑素(Mel)对过氧化氢(H202)氧化损伤的人视网膜色素上皮(hRPE)细胞的保护作用及其作用机制.方法 实验研究.采用600μmoL/L H2O2建立体外培养的hRPE细胞氧化损伤模型.实验分为6组:溶剂对照组、600μmoL/L H2O2+溶剂组(H2O2损伤模型组)、600μmoL/L H2O2+10-7mol/L Mel组、600μmoL/L H2O2+10-6moL/L Mel组、600 μmol/L H2O2+10-5mol/L Mel组、600μmol/L H2O2+10-4moL/L Mel组.通过四甲基偶氮唑盐(MTT))法检测细胞活性;测定细胞内超氧化物歧化酶(SOD)活性和丙二醛(MDA)含量以反映细胞氧化损伤程度;分别用DNA Ladders电泳法和流式细胞仪检测细胞的凋亡情况.溶剂对照组与600μmol/L H2O2组间均数比较采用随机区组设计的t检验;600μmol/L H2O2组以及600μmol/L H2O2+不同浓度Mel组间均数比较采用单因素5水平设计的方差分析,组间两两比较采用LSD-t检验.结果 H2O2模型组较对照组细胞活性明显降低、SOD活件降低、MDA含量增加、凋亡率升高,差异均有统计学意义(t=2.25,39.50,68.42;P<0.05);Mel干预组较模型组细胞活性升高、SOD活性升高、MDA含最减少、凋亡率降低,差异有统计学意义(P<0.05),并与药物浓度的变化呈正相关趋势.结论 Mel对H2O2诱导的RPE的氧化损伤具有保护作用,其机制可能与影响细胞活性、增强抗氧化酶活性、减少细胞凋亡有关.     

褪黑素对体外培养的人视网膜色素上皮细胞氧化损伤的保护作用

Objective To investigate the protective effects of melatonin on the retinal pigment epithelium (RPE) against oxidative damage induced by hydrogen dioxide (H2O2) and its mechanism. Methods The RPE cells were seeded and divided into normol control group, oxidative damage group and the treatment group (treated with melatonin at the concentration of 1×10.7 mol/L,1×10-6 mol/L, 1×10-5 mol/L and 1×10-4 mol/L). The model of oxidative damage on the RPE cells was established by culturing the RPE cells with H2O2 at the concentration of 600 μmol/L for 1 hour in vitro. The cell viability of RPE cells was detected by the methyl thiazolyl tetrazolium (MTT) method. The degree of oxidative damage was evaluated by detecting the superoxide dismutase (SOD) and maleic dialdehyde (MDA). Apoptosis was detected qualitatively using the DNA Ladders electrophoretic mothed, and quantitatively using the Annexin V-FITC/PI double staining flow cytometry. Results Compared with normal control group, the oxidative damage group had low cell viability, low SOD and high MDA contents, and high apoptosis rate(t=2.25,39.50,68.42;P<0.05). Compared with oxidative damage group, the treatment group had high cell viability, high SOD and low M DA contents, and low apoptosis rate (P<0.05). Conclusions Melatonin has a protective effect on the RPE against oxidative damage induced by H2O2. The mechanism may involve in reinforcing the cell viability, strengthening the activity of antioxidase, and reducing the apoptosis.     

褪黑素对体外培养的人视网膜色素上皮细胞氧化损伤的保护作用

Objective To investigate the protective effects of melatonin on the retinal pigment epithelium (RPE) against oxidative damage induced by hydrogen dioxide (H2O2) and its mechanism. Methods The RPE cells were seeded and divided into normol control group, oxidative damage group and the treatment group (treated with melatonin at the concentration of 1×10.7 mol/L,1×10-6 mol/L, 1×10-5 mol/L and 1×10-4 mol/L). The model of oxidative damage on the RPE cells was established by culturing the RPE cells with H2O2 at the concentration of 600 μmol/L for 1 hour in vitro. The cell viability of RPE cells was detected by the methyl thiazolyl tetrazolium (MTT) method. The degree of oxidative damage was evaluated by detecting the superoxide dismutase (SOD) and maleic dialdehyde (MDA). Apoptosis was detected qualitatively using the DNA Ladders electrophoretic mothed, and quantitatively using the Annexin V-FITC/PI double staining flow cytometry. Results Compared with normal control group, the oxidative damage group had low cell viability, low SOD and high MDA contents, and high apoptosis rate(t=2.25,39.50,68.42;P<0.05). Compared with oxidative damage group, the treatment group had high cell viability, high SOD and low M DA contents, and low apoptosis rate (P<0.05). Conclusions Melatonin has a protective effect on the RPE against oxidative damage induced by H2O2. The mechanism may involve in reinforcing the cell viability, strengthening the activity of antioxidase, and reducing the apoptosis.     

褪黑素对体外培养的人视网膜色素上皮细胞氧化损伤的保护作用

Objective To investigate the protective effects of melatonin on the retinal pigment epithelium (RPE) against oxidative damage induced by hydrogen dioxide (H2O2) and its mechanism. Methods The RPE cells were seeded and divided into normol control group, oxidative damage group and the treatment group (treated with melatonin at the concentration of 1×10.7 mol/L,1×10-6 mol/L, 1×10-5 mol/L and 1×10-4 mol/L). The model of oxidative damage on the RPE cells was established by culturing the RPE cells with H2O2 at the concentration of 600 μmol/L for 1 hour in vitro. The cell viability of RPE cells was detected by the methyl thiazolyl tetrazolium (MTT) method. The degree of oxidative damage was evaluated by detecting the superoxide dismutase (SOD) and maleic dialdehyde (MDA). Apoptosis was detected qualitatively using the DNA Ladders electrophoretic mothed, and quantitatively using the Annexin V-FITC/PI double staining flow cytometry. Results Compared with normal control group, the oxidative damage group had low cell viability, low SOD and high MDA contents, and high apoptosis rate(t=2.25,39.50,68.42;P<0.05). Compared with oxidative damage group, the treatment group had high cell viability, high SOD and low M DA contents, and low apoptosis rate (P<0.05). Conclusions Melatonin has a protective effect on the RPE against oxidative damage induced by H2O2. The mechanism may involve in reinforcing the cell viability, strengthening the activity of antioxidase, and reducing the apoptosis.     

黄芪多糖对过氧化氢诱导人视网膜色素上皮细胞氧化损伤的保护作用

目的观察黄芪多糖(astragalus polysaccharide,APS)对过氧化氢诱导的人视网膜色素上皮细胞(retinal pigment epithelium,RPE)氧化损伤的保护作用。方法培养的人RPE细胞系ARPE-19分为正常对照组、过氧化氢损伤组和不同浓度(1000 mg·L-1、500mg·L-1)APS干预组,正常对照组不作任何处理,过氧化氢损伤组加入200μmol·L-1过氧化氢,APS干预组加入不同浓度(1000 mg·L-1、500 mg·L-1)APS后加入200μmol·L-1过氧化氢。用倒置荧光显微镜观察各组细胞形态变化;MTT检测各组细胞活性;DAPI染色观察各组细胞核形态学变化;实时细胞电子分析系统(real-time cell electronic sensing system,RT-CES)实时记录细胞生长状态;应用caspase-3活性检测试剂盒测定各组细胞中caspase-3的活性。结果过氧化氢损伤组细胞皱缩,悬浮细胞增多,APS干预后细胞状态改善。MTT检测结果显示:1000 mg·L-1、500 mg·L-1APS孵育24 h后细胞存活率分别为(99.86±3.64)%和(101.03±3.52)%,与正常对照组(100%)相比,差异均无统计学意义(均为P>0.05),过氧化氢损伤组细胞存活率为(56.49±2.30)%,与正常对照组相比显著降低(P<0.01),1000 mg·L-1、500 mg·L-1APS干预后,细胞存活率升高到(88.14±2.97)%和(80.75±3.13)%,与过氧化氢损伤组相比差异均有显著统计学意义(均为P<0.01)。DAPI染色显示正常细胞核均匀淡染,过氧化氢损伤组出现强荧光点和致密的细胞核,APS处理后凋亡程度减轻。RT-CES显示,APS处理可以减少过氧化氢造成的细胞损伤。Caspase-3检测发现过氧化氢造成细胞caspase-3水平升高,APS处理后caspase-3水平下降。结论 APS可以抑制过氧化氢诱导的人RPE细胞系ARPE-19的凋亡,这为APS的临床应用提供了一定的实验基础。     

人视网膜色素上皮细胞累积性氧化损伤的机制探讨

目的探讨累积性氧化损伤对培养的人视网膜色素上皮（human retinal pigment epithelium,RPE）细胞生长的影响及其影响机制.方法体外培养的人RPE细胞中加入600 μmol/L过氧化氢（hydrogen peroxide,H2O2）,分别培养1、6、12、24、72 h,采用四甲基偶氮唑盐比色（MTT）法检测不同时段H2O2对人RPE细胞生长的影响,应用荧光素标记的连接素V-碘化丙锭（annexin V-fluorescein isothiocyanate-propidium iodium,Av-FITC-PI）双染色流式细胞法测定人RPE细胞凋亡.采用免疫印迹法检测衰老相关蛋白Clusterin的表达.结果（1）MTT法检测结果：600 μmol/L的H2O2作用6 h,即可抑制人RPE细胞生长,H2O2作用时间延长至24 h,其作用时间与人RPE细胞增长抑制率成正比.（2）Av-FITC-PI法检测结果：600 μmol/L的H2O2作用6 h,可诱导人RPE细胞凋亡,且随时间的延长而增加,差异有统计学意义（P＜0.05）;而72 h后则引起细胞的坏死.（3）免疫印迹法检测结果：Clusterin蛋白在正常人RPE细胞呈弱表达,H2O2作用12 h可见Clusterin蛋白表达增强,24 h组表达开始减弱,72 h组仅见微弱表达.结论600μmol/L的H2O2长时间作用可抑制人RPE细胞生长,诱导人RPE细胞凋亡;同时诱导衰老相关蛋白Clusterin的表达;而更长时间的累积损伤则造成人RPE细胞的坏死.H2O2氧化损伤可以模拟人RPE细胞的老年性改变.