H_2O_2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

目的观察以H_2O_2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。

Objective Some 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. Methods Human LECs（SRA01/04） were cultured in DMED medium containing 20% fetal bovine serum for 24 hours and exposed to 1 mmol/L H2O2 for 0 （ control group） ,5,10,15,30 and 60 minutes. The activity of lens cells was detected by MTT（ n = 5 ）. Laser scanning eonfocal 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. Results The 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 H2O2 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 ）. Conclusion Oxidative stress affects the activity of LECs. H2O2 induces redistribution of eaveolin on LECs,indicating eaveolin may participate in intercellular communications.