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

Mei Yu; Zhou Xiyuan

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

更新时间：2023-12-18

- Effect of Slit2 gene transfection on vascular endothelial growth factor in retinal pigment epithelium cells and its mechanism
- Journal of Chongqing Medical University Vol. 38, Issue 11, Pages: 1594-1599(2014)
- 作者机构：
  
  重庆医科大学附属第二医院眼科
- 作者简介：
- 基金信息：
- DOI：
  CLC：
- Published：2014，
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Mei Yu, Zhou Xiyuan. Effect of Slit2 gene transfection on vascular endothelial growth factor in retinal pigment epithelium cells and its mechanism. [J]. Journal of Chongqing Medical University 38(11):1594-1599(2014)
DOI：

Mei Yu, Zhou Xiyuan. Effect of Slit2 gene transfection on vascular endothelial growth factor in retinal pigment epithelium cells and its mechanism. [J]. Journal of Chongqing Medical University 38(11):1594-1599(2014) DOI：

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.

关键词

Slit2基因丝氨酸苏氨酸蛋白激酶细胞外信号调节蛋白激酶血管内皮生长因子

Keywords

Slit2 geneserine-threonine kinaseextracellular signal regulated protein kinasevascular endothelial growth factor

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