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 endothelial 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-threonine 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 assay,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 m RNA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 m RNA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。
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 （P24h=0.002 ,P48h=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 group s （ 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 different 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 PI3K/Akt signaling pathways.
出处
《重庆医科大学学报》
CAS
CSCD
北大核心
2014年第11期1594-1599,共6页
Journal of Chongqing Medical University
基金
国家自然科学基金资助项目(编号:81170858)
