Slit2基因转染对人视网膜色素上皮细胞VEGF表达的影响及其作用机制

Effect of Slit2 gene transfection on vascular endothelial growth factor in retinal pigment epithelium cells and its mechanism

摘要:

目的：研究Slit2基因对人视网膜色素上皮（retinal pigment epithelium，RPE）细胞表达血管内皮生长因子（vascular en－dothelial growth factor，VEGF）的影响，并探讨丝裂原激活（mitogen-activated，MEK）/细胞外信号调节蛋白激酶（extracellular signal regulated protein kinase，ERK）和磷脂酰肌醇-3激酶（phosphatidylinositol 3-kinase，PI3K）/丝氨酸苏氨酸蛋白激酶（serine-threo－nine kinase，AKT）这2条信号通路在此过程中的作用。方法：（1）体外培养人RPE细胞株 RPE-19；（2）将腺病毒介导的Slit2（adenovirus-mediated Slit2，Ad-Slit2）转染RPE细胞，培养12、24 h及48 h；（3）转染前将MEK信号通路阻断剂PD98059、PI3K信号通路阻断剂LY294002预处理RPE细胞1 h；（4）用real-time PCR及酶联免疫吸附试验（enzyme linked immunosorbent as－say，ELISA）检测VEGF的表达水平，用Western blot检测磷酸化ERK、磷酸化AKT的表达情况。结果：Slit2转染的RPE细胞后，real-time PCR结果显示不同时间之间VEGF mRNA表达有差别（F=12.789，P=0.018），组别和时间之间存在交互作用（F=13.375，P=0.017），不同组别有差别（F=16.855，P=0.015），在24 h与48 h，Ad-Ｓlit2组高于空腺病毒组（P24 h=0.002，P48 h=0.018）。ELISA结果显示不同时间之间VEGF 蛋白表达有差别（F=24.886，P=0.000），组别和时间之间存在交互作用（F=23.524，P=0.000），不同组别有差别（F=28.250，P=0.006），在48 h，Ad-Slit2组高于空腺病毒组（P=0.003）。Western blot结果可见p-ERK及p-AKT的蛋白表达水平增加，阻断剂PD98059及LY294002分别能降低ERK、AKT的磷酸化水平，并与Ad-Ｓlit2组相比较，real-time PCR结果显示VEGF mRNA表达水平在不同时间有差别（F=32.202，P=0.000），组别和时间之间存在交互作用（F=8.372，P=0.006）；不同组别存在差别（F=15.062，P=0.001），ELISA 结果显示ＶＥＧＦ蛋白表达水平在不同时间之间有差别（F=90.703，P=0.000），组别和时间之间存在交互作用（F=4.968，P=0.005），不同组别存在差别（F=69.636，P=0.000）。结论：PI3K/AKT以及MEK/ERK这两条信号通路参与了Slit2所诱导的RPE细胞中VEGF表达水平增加。

Abstract:

Objective：To investigate the effect of Slit2 gene on vascular endothelial growth factor（VEGF）in retinal pigment epithelium（RPE）cells and the function of PI3K/AKT and MEK/ERK signaling pathways during the course. Methods：（1）human APRE-19 cells were cultured in vitro. （2）RPE cells were transfected by adenovirus-mediated Slit2（Ad-Slit2），then cultured for 12，24，48 h. （3）Before being transfected by Ad-Slit2，RPE cells were pretreated with MEK inhibitor PD98059 and PI3K inhibitor LY294002 for 1 h. （4）VEGF mRNA expression levels were detected by real-time PCR and the protein expression levels were detected by ELISA. The protein expression levels of p-ERK，p-AKT were detected by Western blot. Results：After RPE cells being transfected with Ad-Slit2，real-time PCR result showed that there were significant statistically differences in VEGF mRNA expression among different time points（F=12.789，P=0.018） and among different groups（F=16.855，P=0.015）；time points and groups were interrelated；at 24 h and 48 h，the VEGF mRNA expression was higher in Ad-slit2 group than in adenovirus group（P24 h=0.002 ，P48 h=0.018）. ELISA results showed that there were significant statistically differences in VEGF protein expression among different time points（F=24.886，P=0.000） and among different groups（F=28.250，P=0.006）；time points and groups were interrelated；at 48 h，the VEGF protein expression was higher in Ad-Slit2 group than in adenovirus group（P=0.003）. Western blot showed that p-ERK and p-AKT protein expressions increased in RPE cells transfected with Ad-Slit2；PD98059 decreased the activation of p-ERK and LY294002 decreased the activation of p-AKT. real-time PCR results showed that there were statistically significant differences in VEGF mRNA expression among different time points（F=32.202，P=0.000） and among different groups（F=15.062，P=0.001）；time points and groups were interrelated（F=8.372，P=0.006）. ELISA results showed there were statistically significant differences in VEGF protein expression among different time points（F=90.703，P=0.000） and among dif－ferent groups（F=69.636，P=0.000）；time points and groups were interrelated（F=4.968，P=0.005）. Conclusion：The expression of the VEGF in RPE cells transfected with Ad-Slit2 can be increased by MEK/ERK and P13K/Akt signaling pathways.