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.
目的 觀察氧化應激對人視網膜色素上皮(RPE)細胞緊密連接屏障功能及緊密連接蛋白occludin、claudin-1 ～4錶達水平的影響.方法 實驗研究.培養人RPE細胞株D407,分為H202處理組和H2O2未處理組(對照組).MTT法觀察不同濃度H2O2對D407細胞活性的影響；分彆應用經上皮電阻(TER)和熒光素鈉滲透實驗檢測低濃度H2O2作用24h和72 h後RPE緊密連接屏障功能的變化；通過實時熒光定量PCR法和免疫印跡法分彆從mRNA和蛋白水平檢測H2O2作用24h後對緊密連接蛋白occludin 、claudin-1 ～4錶達水平的影響.採用t檢驗統計分析TER、熒光素鈉滲透實驗、實時熒光定量PCR法、免疫印跡法的檢測結果,採用單因素方差分析統計分析細胞活力結果.結果 H2O2≤0.4 mmol/L時,處理24h對RPE細胞的活力無明顯影響.選擇0.2 mmol/L為後序試驗的濃度.D407細胞培養8d後,TER達到穩定狀態.0.2 mmol/L H2O2作用3h後TER開始下降,至12 h齣現明顯差異(18.62±1.89比24.11±0.96,t=4.490,P=0.013),24h接近最大效應(11.86±1.19比24.13±1.26,t=12.260,P=0.000),維持至72 h(11.56±1.47比24.33±1.52,t=10.460,p=0.000).H2O2處理D407細胞24 h後加入熒光素鈉,不同時間點處理組的熒光滲透百分比均高于對照組(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處理D407細胞24h後,claudin-1、3、4的mRNA和蛋白錶達水平較對照組下調(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和蛋白錶達水平則較對照組明顯上調(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和蛋白錶達水平在處理組和對照組的差異均無統計學意義(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).結論 氧化應激能夠破壞RPE緊密連接的完整性,使其屏障功能受損,而claudin-1～4的錶達水平變化可能在RPE的氧化損傷中髮揮重要的作用.
목적 관찰양화응격대인시망막색소상피(RPE)세포긴밀련접병장공능급긴밀련접단백occludin、claudin-1 ～4표체수평적영향.방법 실험연구.배양인RPE세포주D407,분위H202처리조화H2O2미처리조(대조조).MTT법관찰불동농도H2O2대D407세포활성적영향；분별응용경상피전조(TER)화형광소납삼투실험검측저농도H2O2작용24h화72 h후RPE긴밀련접병장공능적변화；통과실시형광정량PCR법화면역인적법분별종mRNA화단백수평검측H2O2작용24h후대긴밀련접단백occludin 、claudin-1 ～4표체수평적영향.채용t검험통계분석TER、형광소납삼투실험、실시형광정량PCR법、면역인적법적검측결과,채용단인소방차분석통계분석세포활력결과.결과 H2O2≤0.4 mmol/L시,처리24h대RPE세포적활력무명현영향.선택0.2 mmol/L위후서시험적농도.D407세포배양8d후,TER체도은정상태.0.2 mmol/L H2O2작용3h후TER개시하강,지12 h출현명현차이(18.62±1.89비24.11±0.96,t=4.490,P=0.013),24h접근최대효응(11.86±1.19비24.13±1.26,t=12.260,P=0.000),유지지72 h(11.56±1.47비24.33±1.52,t=10.460,p=0.000).H2O2처리D407세포24 h후가입형광소납,불동시간점처리조적형광삼투백분비균고우대조조(20 min:25％±3％비12％±4％,t=-4.50,P=0.011；40 min:36％±4％비16％±5％,t=-5.41,P=0.006;60 min:51％±5％비29％±6％,t=-4.88,P=0.008).H2O2처리D407세포24h후,claudin-1、3、4적mRNA화단백표체수평교대조조하조(claudin-1 mRNA:0.98±0.18비0.28±0.12,t=5.60,P=0.005,claudin-1단백:48±10비100±12,t =5.77,P=0.004；claudin-3mRNA:0.37±0.12비1.03±0.15,t =5.95,P=0.004,claudin-3단백:63±13비100±15,t=3.23,P =0.032:claudin-4 mRNA:0.38±0.11비0.99±0.17,t=5.22,P=0.002,claudin-4단백:57±12비100±13,t=4.21,P=0.014),claudin-2 mRNA화단백표체수평칙교대조조명현상조(mRNA:1.01±0.22비3.96±0.24,t=-15.69,P=0.000,단백:195±15비100±13,t=-8.29,P=0.001),단occludin적mRNA화단백표체수평재처리조화대조조적차이균무통계학의의(mRNA:1.30±0.21비1.02±0.16,t=-1.84,P=0.140,단백:109±15비100±14,t=-0.76,p=0.490).결론 양화응격능구파배RPE긴밀련접적완정성,사기병장공능수손,이claudin-1～4적표체수평변화가능재RPE적양화손상중발휘중요적작용.
Objective 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.
