背景 活性氧中间产物可导致细胞氧化损伤,视网膜色素上皮(RPE)细胞对视细胞外节膜盘的吞噬产生了大量的活性氧中间产物过氧化氢(H2O2),易对RPE细胞本身造成反复的氧化损伤. 目的 探讨非致死性H2O2持续性损伤对RPE细胞连接的影响. 方法 ARPE-19细胞株以8×104个/L的密度接种在96孔板中,在24孔板中细胞爬片的接种密度为4×104个/L,并在DMEM/F12培养液中培养,无血清培养液饥饿培养24 h后用于实验.将不同浓度(0～0.60 mmol/L)的H2O2加入培养液,单溶液细胞增生(MTS)法检测不同浓度H2O2持续性损伤对RPE细胞活力的影响；用Transwell小室法培养细胞,跨上皮细胞电阻(TER)法检测细胞单层的形成时间；以罗丹明-异硫氰酸荧光素标记葡聚糖的量测定H2O2对RPE细胞单层通透性的影响；免疫荧光法检测RPE细胞之间紧密连接蛋白ZO-1的表达情况.结果 不同质量浓度H2O2作用组细胞活力(A490值)的差异有统计学意义(F=991.501,P=0.000),0.20～0.60 mmol/L H2 02组细胞活力明显低于对照组,差异均有统计学意义(t=3.200、11.368、4.256、2.929、10.818、15.433、64.125、98.805,P＜0.05).ARPE-19细胞接种于Transwell小室后l1d时TER值为(24.9±1.3)Ω ·cm2,7d时为(17.8±1.4)Ω·cm2,差异有统计学意义(t=5.228,P=0.014)；至第15天左右达到稳定水平,约为(25.9±0.9)Ω·cm2.细胞通透性检测表明,无细胞的空白组葡聚糖渗漏量(相对荧光强度)为433.08±51.53,无H2O2的对照组渗漏量为255.39±16.44,明显低于空白组,差异有统计学意义(t=12.515,P=0.006)；H2O2处理的实验组渗漏量为363.28±26.17,较对照组渗漏增加,差异亦有统计学意义(t=14.412,P=0.005).免疫荧光检测可见无H2O2对照组细胞ZO-1连接完整,H2O2组较无H2O2对照组细胞ZO-1连接呈断裂状. 结论 非致死性H2O2持续性损伤对RPE细胞连接有破坏作用.%Background Reactive oxygen intermediate products lead to the oxidative injury of cells.Retinal pigment epithelial(RPE) cells produce lots of reactive oxygen intermediate products during the swallow of out disc,but how this procedure cause the persistent oxidative injury of RPE cells is poorly understood. Objective The present study was to evaluate the effect of non-lethal H2 O2 -induced persistent oxidative injury on RPE barrier in vitro.Methods ARPE-19 cell links were inoculated on 96 well plate at the density of 8×104 cells/L and the cell climbing slice of 24 well at the density of 4× 104 cells/L.The cells were cultured in DMEM/F12 medium,and the cells cultured for 24 hours in free-serum medium were used in the experiment.0-0.6 mmol/L of H2O2 were added into the medium.Cellular viability was assessed using 3- ( 4,5-dimethylthiazol-2-yl ) -5- ( 3-carboxymethoxyphenyl ) -2- ( 4-sulfophenyl ) 2H-tetrazolium(MTS) assays.Transepithelial electrical resistance (TER) was used to detect cell monolayer forming time after culture in Trsnswell chamber.The permeability of cell monolayer was examined by rhodamine isothiocyanate-dextran transepithelial flux,and immunofluorescence was used to investigate the distribution of the junction protein zonula occludens (ZO-1). Results The total difference was found in the cell vitality(A490) among the different concentrations of H2 O2 ( F =991.501,P =0.000 ).Compared with 0 mmoL/L H2 O2 group,the A490 values was gradually lowed from 0.20 mmol/L H2O2 group to 0.60 mmol/L H2O2 group (P ＜ 0.05 ).H2O2 at the concentrations of ＞0.20 mmol/L lowed the viability of RPE cells.The TER value was ( 24.9 ± 1.3 ) Ω · cm2 in 11 days,( 17.8± 1.4)Ω · cm2 in 7 days after inoculation on transwell chamber,showing a significant difference between them (t=5.228,P=0.014).RPE formed the stable tight junction on day 15 with the TER value (25.9±0.9 ) Ω · cm2.The leakage amount ( relative fluorescence intensity ) of the dextran was 255.39 ± 16.44 in non-H2 O2 control group,exhibiting a significant lowing in comparison with free-cell blank group (433.08±51.53)( t =12.515,P =0.006 ),and that of H2 O2 group was significant increased in comparison with non-H2 O2 control group ( t =14.412,P=0.005).Immunofluyorescence assay showed intact intercellular ZO-1 junction in non-H2O2 control group,but the breakage of ZO-1 junction was seen in H2O2 group. Conclusions The results indicate that non-lethal H2O2 can destroy RPE barrier and further lead to the persistent oxidative injury of RPE cells.
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