EGF通过PI3K/AKT通路诱导人晶状体上皮细胞移行

Human lens epithelial cells migration induced by epidermal growth factor through PI3K/AKT pathways

目的研究表皮生长因子(epidermal growth factor,EGF)诱导人晶状体上皮细胞移行及其信号通路机制。方法培养人晶状体上皮细胞,用不同浓度EGF处理24h后,Phagokinetic Track Motility分析细胞的移动性;EGF(100μg·L-1)孵育细胞不同时间后,Westernblot法检测磷酸化EGFR和AKT,并分别用PD153035(EGFR激酶抑制剂)和LY294002(PI3K/AKT抑制剂)预孵育细胞,观察上述指标;EGF(100μg·L-1)孵育细胞不同时间(12h、24h、48h)后,用酶谱法分析基质金属蛋白酶-2(matrix metallopreteinase-2,MMP-2),并用PD153035、LY294002预孵育细胞,检测MMP-2活性及细胞的移行。设立对照组,结果进行单因素方差分析,P<0.05表明差异有统计学意义。结果EGF可诱导培养人晶状体上皮细胞移行,并随浓度增加细胞的移动性有显著性提高;EGF可诱导EG-FR和AKT磷酸化,EGF处理后5min,EGFR和AKT的磷酸化达到峰值,作用可持续1h,EGFR抑制剂可阻断EGF诱导的AKT磷酸化;EGF可显著提高细胞中MMP-2的活性,并随时间延长活性增强,24～48h达到较高水平(1.4倍),PD153035、LY294002可抑制MMP-2的活性以及细胞的移行。结论在人工培养的人晶状体上皮细胞中,EGFR/PI3K/AKT通路介导了EGF刺激MMP-2的激活和体外培养人晶状体上皮细胞的移行,这一通路可被EGFR激酶抑制剂、PI3K/AKT抑制剂阻断,这些涉及的信号通路可能形成潜在的治疗后囊膜混浊的靶点。

Objective To study the cultured human lens epithelial cells （HLECs） migration induced by epidermal growth factor （EGF） and its signal pathway（ PI3K/AKT pathways） mechanism. Methods HLECs were cultured and treated with EGF at concentrations of 1μg·L^-1 ,10 μg·L^-1,50 μg·L^-1,100μg·L^-1 and 200 μg·L^-1. Cell migration was digitized by Phagokinetic Track Motility at 24 hours after EGF treatment. After incubated with 100 μg·L^-1 of EGF, the phosphorylation of EGFR and AKT induced by EGF was analyzed by Western blot at 5 minutes,15 minutes,30 minutes,60 minutes and 120 minutes, then HLECs were pre-incubated with PD153035 （EGFR inhibitor） and LY294002 （PI3K/AKT inhibitor） for two hours, the above-mentioned indexs were observed. After incubated with 100μg·L^-1 of EGF, matrix metalloproteinase-2 （MMP-2） was analyzed by zymography at 12 hours, 24 hours, 48 hours, then HLECs were pre-incubated with PD153035 and LY294002, the activity of MMP-2 and cell migration were examined. The control group was set up ,statistical analysis of the data between the control and treated groups was performed by oneway ANOVA. Values of P 〈 0.05 were considered as significant. Results EGF could induce the cell migration in cultured HLECs in a dose dependent manner. EGF could induce the phosphorylation of EGFR and AKT, which peaked at 5 minutes after EGF treatment, and lasted for 1 hour. EGFR and AKT phosphorylation induced by EGF were inhibited by PD153035 and LY294002, respectively. Furthermore, EGF could increase the activity of MMP-2 in a time dependent manner in HLECs, which peaked between 24 hours to 48 hours after treatment. The activity of MMP-2 and cell migration were inhibited by PD153035 and LY294002. Conclusion EGFR/PI3K/AKT pathway mediated MMP-2 activation and cell migration stimulated by EGF in cultured HLECs in vitro, and this cell signaling pathway can be inhibited by EGFR inhibitor and PI3K/AKT inhibitor, which may be used as potential therapeutic targets in the treatment of posterior capsular opacitication.