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.
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