摘要
目的 探讨白细胞介素（IL）-17F及IL-17受体C（IL-17RC）对大鼠肺微血管内皮细胞（PMVEC）小窝蛋白1（Cav-1）表达的影响.方法 将体外培养的大鼠PMVEC分为时效实验组以及IL-17RC信号通路干预实验组;根据时效实验组结果：即IL-17F诱导大鼠PMVEC的Cav-1及其磷酸化表达峰值的时间,制定IL-17RC信号通路干预实验组IL-17F与大鼠PMVEC孵育时间：（1）时效实验组：100 ng/ml的IL-17F刺激0、0.5、1.5、3.0、6.0、12.0、24.0 h后Western印迹法检测Cav-1表达以及0、10、30、60、90、120 min后检测Cav-1及其磷酸化（p-Cav-1）的表达;（2） IL-17RC信号通路干预实验组：siRNA病毒与大鼠PMVEC预孵育3d后分成2组,第1组给予100 ng/ml的IL-17F刺激60 min后检测p-Cav-1表达,第2组给予100 ng/ml的IL-17F刺激24.0 h后检测Cav-1表达,各组分别设对照组、无意义-siRNA组、IL-17RC-siRNA组、IL-17F组以及无意义-siRNA＋IL-17F组作为参照.结果 IL-17F时间依赖性增强大鼠PMVEC表达Cav-1,0、0.5、1.5、3.0、6.0、12.0、24.0 h相对表达量分别为（1.139 ±0.134）、（1.276±0.166）、（1.604±0.080）、（2.115 ±0.231）、（2.763±0.226）、（3.309±0.493）、（3.963±0.169）,1.5h时开始显著高于0h,24.0h时达峰值且显著高于0、0.5、1.5、3.0、6.0、12.0h（均P＜0.05）;IL-17F时间依赖性增强p-Cav-1表达,0、10、30、60、90、120 min相对表达量分别为（0.540±0.085）、（0.880±0.084）、（1.437±0.297）、（1.491 ±0.212）、（1.017 ±0.210）、（0.882±0.074）,10 min时开始显著高于0 min,60 min时达峰值且显著高于0、10、30、90、120 min（均P＜0.05）,然后逐渐下降,120 min时仍高于0 min.IL-17RC-siRNA可显著抑制IL-17F诱导的Cav-1和p-Cav-1的表达（2.126±0.318比3.897±0.424和1.014±0.136比1.431±0.298,均P＜0.05）.结论 IL-17F时间依赖性增加大鼠PMVEC的Cav-1及p-Cav-1表达;沉默大鼠PMVEC的IL-17RC表达显著抑制IL-17F诱导的Cav-1及p-Cav-1表达.
Objective To explore the expression of caveolin-1 （Cav-1） induced by interleukin-17F/IL-17 receptor C （IL-17F/IL-17RC） in rat pulmonary microvascular endothelial cells （PMVECs）.Methods Cultured PMVECs were divided into two groups of time-dependent experiment and IL-17RC signal pathway intervention according to the results of time-dependent experimental group：namely peak time of IL-17F-induced Cav-1 and its phosphorylation （p-Cav-1） expression of PMVEC for formulating the incubation time between IL-17F and PMVEC in IL-17RC signal pathway intervention group.Time-dependent experiment group：Western blot was used to detect the expression of Cav-1 after IL-17F stimulation for 0,0.5,1.5,3.0,6.0,12.0,24.0 h.Cav-1 and its phosphorylation expression after IL-17F challenge for0,10,30,60,90,120 min were evaluated by Western blot.IL-17RC signal pathway intervention group：PMVECs were divided into two groups after a 3-day pre-treatment of siRNA.The first group received a 60-min stimulation of 100 ng/ml IL-17F before detecting the expression of p-Cav-1 while the second group was subject to a 24-hour stimulation of 100 ng/ml IL-17F before detection.In addition,control,meaningless-siRNA,IL-17RC-siRNA,IL-17F and meaningless-siRNA ＋ IL-17F groups were set as references for two groups respectively.Results IL-17F up-regulated the expression of Cav-1 in a time-dependent manner.At 0,0.5,1.5,3.0,6.0,12.0,24.0 h,the relative expression levels of Cav-1 were （1.139 ±0.134）,（1.276±0.166）,（1.604 ±0.080）,（2.115 ±0.231）,（2.763 ±0.226）,（3.309 ±0.493） and （3.963 ± 0.169）.At 1.5 h,it was significantly higher than 0 h,peaked at 24.0 h and remained significantly higher than 0,0.5,1.5,3.0,6.0,12.0 h （all P 〈 0.05）.And IL-17F increased the expression of p-Cav-1 in a time-dependent manner.At 0,10,30,60,90,120 min,the relative expression levels of p-Cav-1 were （0.540 ± 0.085）,（0.880 ± 0.084）,（1.437 ± 0.297）,（1.491 ± 0.212）,（1.017 ±0.210） and （0.882 ±0.074）.At 10 min,p-Cav-1 was significantly higher than 0 min,peaked at 60 min,remained significantly higher than 0,10,30,90,120 min （all P 〈 0.05） and gradually decreased.At 120 min,it was still higher than 0 min.Compared with IL-17F group,IL-17RC-siRNA significantly inhibited IL-17F-induced Cav-1 and its phosphorylation （2.126 ± 0.318 vs 3.897 ± 0.424 and 1.014 ±0.136 vs 1.431 ±0.298,all P 〈0.05）.Conclusions IL-17F up-regulates the expressions of Cav-1 and p-Cav-1 in a time-dependent manner in PMVECs.And the silenced expression of IL-17RC in PMVECs by IL-17RC-siRNA significantly inhibits the IL-17F-induced expressions of Cav-1 and p-Cav-1.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2015年第12期912-916,共5页
National Medical Journal of China
基金
河北省科技支撑计划（13277746D）
