摘要目的 观察氧化应激对人视网膜色素上皮(RPE)细胞紧密连接屏障功能及紧密连接蛋白occludin、claudin-1 ～4表达水平的影响.方法 实验研究.培养人RPE细胞株D407,分为H202处理组和H2O2未处理组(对照组).MTT法观察不同浓度H2O2对D407细胞活性的影响；分别应用经上皮电阻(TER)和荧光素钠渗透实验检测低浓度H2O2作用24h和72 h后RPE紧密连接屏障功能的变化；通过实时荧光定量PCR法和免疫印迹法分别从mRNA和蛋白水平检测H2O2作用24h后对紧密连接蛋白occludin 、claudin-1 ～4表达水平的影响.采用t检验统计分析TER、荧光素钠渗透实验、实时荧光定量PCR法、免疫印迹法的检测结果,采用单因素方差分析统计分析细胞活力结果.结果 H2O2≤0.4 mmol/L时,处理24h对RPE细胞的活力无明显影响.选择0.2 mmol/L为后序试验的浓度.D407细胞培养8d后,TER达到稳定状态.0.2 mmol/L H2O2作用3h后TER开始下降,至12 h出现明显差异(18.62±1.89比24.11±0.96,t=4.490,P=0.013),24h接近最大效应(11.86±1.19比24.13±1.26,t=12.260,P=0.000),维持至72 h(11.56±1.47比24.33±1.52,t=10.460,p=0.000).H2O2处理D407细胞24 h后加入荧光素钠,不同时间点处理组的荧光渗透百分比均高于对照组(20 min:25％±3％比12％±4％,t=-4.50,P=0.011；40 min:36％±4％比16％±5％,t=-5.41,P=0.006;60 min:51％±5％比29％±6％,t=-4.88,P=0.008).H2O2处理D407细胞24h后,claudin-1、3、4的mRNA和蛋白表达水平较对照组下调(claudin-1 mRNA:0.98±0.18比0.28±0.12,t=5.60,P=0.005,claudin-1蛋白:48±10比100±12,t =5.77,P=0.004；claudin-3mRNA:0.37±0.12比1.03±0.15,t =5.95,P=0.004,claudin-3蛋白:63±13比100±15,t=3.23,P =0.032:claudin-4 mRNA:0.38±0.11比0.99±0.17,t=5.22,P=0.002,claudin-4蛋白:57±12比100±13,t=4.21,P=0.014),claudin-2 mRNA和蛋白表达水平则较对照组明显上调(mRNA:1.01±0.22比3.96±0.24,t=-15.69,P=0.000,蛋白:195±15比100±13,t=-8.29,P=0.001),但occludin的mRNA和蛋白表达水平在处理组和对照组的差异均无统计学意义(mRNA:1.30±0.21比1.02±0.16,t=-1.84,P=0.140,蛋白:109±15比100±14,t=-0.76,p=0.490).结论 氧化应激能够破坏RPE紧密连接的完整性,使其屏障功能受损,而claudin-1～4的表达水平变化可能在RPE的氧化损伤中发挥重要的作用.

abstractsObjective To investigate the effects of hydrogen peroxide ( H2 O2 ) on the barrier function and expression of tight junction protein in human retinal pigment epithelium (RPE) cells.Methods Experimental study.The human RPE cell line (D407) were cultured and treated with ( H2O2 treated group) or without H2O2 ( normal control group).The effect of H2O2 on cell viability of RPE cells was determined by MTT test.After treated with low concentration of H202 for 24 h to 72 h,transepithelial electrical resistance (TER) of confluent RPE cells was measured by epithelial voltmeter.The permeability of RPE cells to sodium fluorescein was measured.The expressions of the occludin and claudin-1 to -4 were determined by real-time polymerase chain reaction and Western blot analysis.t-text and one-way ANOVA were used to assess statistical significance btween H2O2 treated and normal coutrol groups.Results H2O2 at 0.2 mmol/Lshowed no decrease of cell viability of D407 cells,and this concentration was selected for the present study.The TER of D407 cells gradually increased,peaking at day 8 and then remained stable for 1 week.As compared to the control group,a reduction in the TER was first evident after 3 hours of treatment.Continuous culturing of cells for longer periods further reduced the TER,with a maximum effect after 24 hours of treatment and was maintained to 72 hours (24 h:11.86 ± 1.19 vs.24.13 ± 1.26,t =12.260,P =0.000;72 h:11.56 ± 1.47 vs.24.33 ± 1.52,t =10.460,P =0.000 ).At any time point after adding sodium fluorescein,the permeability values of cells after treated with H2O2 for 24 hours were significantly higher than those of cells without H2O2 treatment (20min:25％ ±3％ vs.12％ ±4％,t =-4.50,P =0.011 ;40 min:36％ ±4％ vs.16％ ±5％,t =-5.41,P =0.006;60 min:51％ ±5％ vs.29％ ±6％,t =-4.88,P =0.008 ).The expression of mRNA and protein in claudin-1,-3,and -4 were all downregulated in D407 cells treated with H2O2,whereas the expression of claudin-2 was upregulated ( claudin-1 mRNA:0.98 ± 0.18vs.0.28 ±0.12,t =5.60,P =0.005,claudin-1 protein,48 ± 10 vs.100 ± 12,t =5.77,P =0.004;claudin-3mRNA:0.37 ±0.12 vs.1.03 ±0.15,t =5.95,P =0.004;claudin-3 protein:63 ± 13 vs.100 ± 15,t =3.23,P=0.032;claudin-4 mRNA:0.38 ±0.11 vs.0.99 ±0.17,t =5.22,P=0.002,claudin-4 protein,57 ± 12vs.100 ± 13,t =4.21,P =0.014 ).However,the expression of these occludins did not differ bettween cells treated with and without H2O2(mRNA:1.30 ±0.21 vs.1.02 ±0.16,t=- 1.84,P =0.140;protein:109 ±15 vs.100 ± 14,t =-0.76,P =0.490 ).Conclusion Oxidative stress causes increase in the paracellular permeability of RPE cells in vitro,which may depends on the changes in expression of certain transmembrane proteins associated with the tight junction.