| ObjectiveAfter-cataract, also called posterior capsular opacification (PCO), is the most common postoperative complication of modern cataract surgery, and which can cause obstruction in vision again. After cataract surgery, PCO develops because of the proliferation, transdifferentiation and differentiation with extra cellular matrix secretion of residual lens epithelial cells (LECs), the most key is proliferation of lens epithelial cells. For many years, many scholars of domestic and overseas had been committed to study the methods to suppress the proliferation of lens epithelial cells by drugs.Currently, the hot research field of cell proliferation is the signal transduction pathways, phosphatidyinositol 3-kinase/protein kinase B(PI3K/AKT) signaling pathway has an important role in growth, survival, proliferation and apoptosis of cells, and LY294002 as the specific inhibitor of signaling pathways, can inhibit cell proliferation. S phase kinase associated protein 2, as the control factor, can mediate the regulation of cell cycle and cell proliferation. This study applies PI3K inhibitor LY294002 to lens epithelial SRA01/04 cell line in vitro culture, to observe the effect on cell proliferation and study the possible mechanism, which provide theoretical basis for prevention and treatment of after-cataract in clinic.Methods1. Experimental objectsHuman lens epithelial SRA01/04 cell line was purchased from Chinese Academy of Sciences, Beijing.2. Methods (1) Cell Culture:SRA01/04 cells were cultured in RPMI1640 medium with 10% fetal calf serum, and the cells were maintained at 37℃in a humidified atmosphere of 5% CO2 in air.(2) Cells were divided into two groups:the control group and the experimental group. PI3K/AKT pathway specific inhibitor LY294002 was added to the experimental group at 25μmol/L. The same amount volume of DMSO was added to the control group.(3) MTT assay was used to detect the effect of LY294002 on cell proliferation. The growth curves were drawn, and the appropriate length of treated time was optimized.(4) Flow cytometer (FCM) was used to analyze the cell cycle progression in SRA01/04 cell line treated with DMSO or LY294002.(5) Western-blot was used to detect the expression level of Skp2 protein in SRA01/04 cell line treated with DMSO or LY294002.(6) Real-time RT-PCR was used to detect the mRNA level in SRA01/04 cell line treated with DMSO or LY294002.3. Statistical AnalysisStatistical software SPSS 11.5 was usd for t test of differences between the two groups, and the results are presented as mean±SD. P≤0.05 was considered statistically significant.Results1. Inhibition of cell proliferation by PI3K Inhibitor LY294002 in human lens epithelial cellsMTT assays showed that the value of experimental absorbency was lower than the control group, the difference was statistically significant decline (P＜0.05). The rate of cell growth slowed, at 12,24,36 hours, the inhibition rate of cell growth was 21.4%,33.8% and 24.2%, respectively. 2. G1/S cell cycle arrest by LY294002 in human lens epithelial cellsFCM analysis showed that the cells in G1 phase treated with LY294002 increased from (56.73±1.35)% to (66.15±2.63)%, while the cells in S phase decreased from (37.32±5.54)% to (27.34±6.58)%. The differences were statistically significant (P＜0.05).3. The effect of LY294002 on expression of Skp2 proteinWestern blot showed that compared to DMSO treated control group, there was an decrease expression of Skp2 protein in SRA01/04 cells. The differences were statistically significant (P＜0.05).4. The effect of LY294002 on the expression of Skp2 mRNAAs shown in Real-time RT-PCR, the expression level of Skp2 mRNA was decreased in SRA01/04 cells. The differences were statistically significant (P＜0.05).Conclusions1. This study demonstrated a role for PI3K inhibitor LY294002 in the prevention of cell proliferation in human lens epithelial SRA01/04 cell line.2. The expression level of Skp2 was decreased by LY294002, and the cell cycle progression was limited at the G1/S transition.
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