PI3K抑制剂LY294002对人晶状体上皮细胞中S期激酶相关蛋白2表达的影响

目的探讨PI3K抑制剂LY294002对体外培养的人晶状体上皮细胞（LECs）增生和细胞周期S期激酶相关蛋白2（Skp2）表达的影响。方法 MTT比色法检测25μmol/L的LY294002处理人LECs细胞株SRA01/04后12h和24h细胞的增生情况;流式细胞仪检测LY294002作用24h后细胞周期的分布情况;Westernblot和real-timePCR法分别测定LY294002处理24h后细胞中Skp2及其mRNA的表达情况。结果 25μmol/L的LY294002作用于SRA01/04细胞0～24h,SRA01/04细胞的增生受抑制,且作用24h时对SRA01/04细胞的抑制作用最明显。LY294002组中处于G1期和S期的细胞占总细胞数的百分率分别为（66.15±2.63）%和（27.34±6.58）%,对照组为（56.73±1.35）%和（37.32±5.54）%,LY294002组细胞Skp2及其mRNA的表达量明显减少。结论 PI3K抑制剂LY294002可抑制人LECs的增生,使部分细胞停滞于细胞周期的G1期,LY294002抑制人LECs的增生可能与Skp2的表达下调有关。

Effects of PI3K inhibitor LY294002 on expression of S-phase kinase associated protein 2 in human lens epithelial cells

Background The proliferation of residual human lens epithelial cells （LECs） after cataract surgery leads to the formation and development of posterior capsular opacification （PCO）.Although a variety of studies have increased our understanding of the pathogenesis of PCO,the cellular mechanism underlying the development of the PCO is still unclear.Objective This study attempted to characterize the effect of PI3K inhibitor,LY294002,on the proliferation of human lens epithelial cells （LECs） and the expression of S-phase kinase associated protein 2 （Skp2） in vitro.Methods Human LECs strains （SRA01/04） were cultured in RPMI1640 medium containing 10% fetal bovine serum.25 μmol/L LY294002 with DSMO was added into the medium after 24 hours.Only the same volume of DSMO was used as control group.The inhibiting rate of LY294002 on the proliferation of human LECs was detected in 12 and 24 hours after addition of LY294002 using MTT assay.Expressions of Skp2 protein and mRNA in cultured LECs were detected using Western blot and real-time PCR in 24 hours after treatment of 25 μmol/L LY294002 respectively.The effect of LY294002 （25 μmol/L） on LECs cellular cycle was analyzed by flow cytometry.Results The A490 values of LECs were quite different between DSMO group and 25 μmol/L LY294002 group in both 12 hours and 24 hours after addition of LY294002 （t=4.899,P0.01;t=4.879,P0.01 respectively）.Proliferation of LECs was significantly declined in 25 μmol/L LY294002 group compared with only DSMO group in both 12 hours and 24 hours （P0.01）.In 24 hours after treatment of 25 μmol/L LY294002,both Skp2/β-actin value and Skp2/GAPDH value were decreased in comparison with only DSMO group （t=26.931,P0.01;t=5.17,P0.01 respectively）.The percentage of LECs in G1 phase was significantly increased in 25 μmol/L LY294002 group compared normal control group （56.73%±1.35% vs 66.15%±2.63%） （t=5.605,P0.01）,and that in S phase was evidently decreased in 25 μmol/L LY294002 group compared normal control group （37.32%±5.54% vs 27.34%±6.58%） （t=4.080,P0.05） in 24 hours after treatment of 25 μmol/L LY294002.Conclusion LY294002 can inhibit the proliferation of human LECs in vitro and arrest LECs to enter into S phase from G1 phase.These effects of LY294002 on LECs may associated with down-regulation of Skp2 expression.