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H₂O₂对小窝蛋白在人晶状体上皮细胞中分布与表达的影响

Effects of hydrogen peroxide on distribution and expression of caveolin in human lens epithelial cells

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【Author】 Jin Hongying ,Yao Ke,Ma Jin,Yang Chunhu,Li Hongwu,Tang Xiajing.Eye Center,Affiliated Second Hospital,College of Medicine of Zhejiang University,Hangzhou 310009,China

【机构】浙江大学医学院第二附属医院眼科中心；浙江大学医学院第二附属医院眼科中心；浙江大学医学院生物电磁试验室；

【摘要】目的观察以H₂O₂刺激晶状体上皮细胞（LECs）后对细胞活性的影响及小窝蛋白（caveolin）在LECs膜及胞浆内的分布和表达量的改变。方法用1mmol/LH2O2刺激LECs不同时间,MTT法观察细胞活性;激光共聚焦显微镜观察caveolin在细胞膜和胞浆内的分布;Western-blot法检测caveolin表达量的变化。结果H2O2作用时间越长,细胞活性越低;对照组与试验组的差异有显著统计学意义。激光共聚焦显微镜观察到人LECs有丰富的caveolin;无刺激时主要分布在膜上;刺激后在膜及胞浆内的分布增多。Western-blot法发现H2O2作用后,caveolin的表达量减少。结论H2O2对人LECs的活性产生影响,并导致细胞caveolin重新分布、表达量减少。更多还原

【Abstract】 ObjectiveSome researches demonstrated that caveolae exist in lens of human and animal.Caveolin is a marker of caveolae.We studied the activity of the human lens epithelial cells （LECs） treated by hydrogen peroxide （H2 O2 ） and observed the distribution and expression of caveolin in LECs membrane and cytoplasm.MethodsHuman LECs（SRA01/04） were cultured in DMED medium containing 20% fetal bovine serum for 24 hours and exposed to 1 mmol/L H2 O2 for 0（control group）,5,10,15,30 and 60 minutes.The activity of lens cells was detected by MTT（n=5）.Laser scanning confocal microscopy was used to observe the distribution of caveolin in cellular membrane and cytoplasm （n=34）.The changes of the expression of caveolin was observed with Western blot analysis （n=7）.SPSS 12.0 was used for statistic analysis.ResultsThe activity of LECs was markedly lowed under the condition of oxidative stress with the time lapse and exhibited a significant difference between the control group and H2 O2 stimulation groups （F=185.984,P<0.01）.Under laser scanning confocal microscopy,abundance caveolin located in cellular membrane and cytoplasm of LECs.The fluorescence intensity on the cells membrane or in cytoplasm was significant increased in H2 O2 stimulation groups in comparison with control group（F=6.196,P<0.01;F=9.009, P<0.01 ）.The cellular membrane of LECs began to break 1 hour after H2 O2 treatment;whereas the caveolin could be still observed.Western blot analysis revealed that the expression level of caveolin was decreased under the oxidative stress compared with control group （F=6.152,P<0.05）.ConclusionOxidative stress affects the activity of LECs. H₂O₂ induces redistribution of caveolin on LECs,indicating caveolin may participate in intercellular communications.更多还原