生物钟蛋白CRY1抑制AngⅡ诱导的小鼠主动脉平滑肌细胞表型蛋白标志物变化  

Circadian clock protein CRY1 inhibits AngⅡ-induced phenotypic transformation of mouse aorta smooth muscular cells

作　　者：岳秀青 宋其泰 刘超利 李桑柔 杨帆 陈睦虎 钟武 YUE Xiuqing;SONG Qitai;LIU Chaoli;LI Sangrou;YANG Fan;CHEN Muhu;ZHONG Wu(Emergency Intensive Care Unit,the Affiliated Hospital of Southwest Medical University,Luzhou 646000;Luzhou People's Hospital,Luzhou 646000;Sichuan Provincial Rehabilitation Hospital,Chengdu 610000,China)

机构地区：[1]西南医科大学附属医院急诊医学部,四川泸州646000 [2]泸州市人民医院,四川泸州646000 [3]四川省康复医院,四川成都610000

出　　处：《基础医学与临床》2023年第4期588-595,共8页Basic and Clinical Medicine

基　　金：西南医科大学附属医院博士科研启动基金(19085);四川省卫生健康委员会医学科技项目(21PJ199)。

摘　　要：目的探讨生物钟(circadian clock)相关蛋白隐花色素1(CRY1)在血管紧张素Ⅱ(AngⅡ)诱导的小鼠主动脉血管平滑肌细胞(VSMCs)表型转化中的作用。方法将VSMCs分为对照(control)组和实验组,用不同浓度AngⅡ处理VSMCs不同时间,RT-PCR和Western blot检测收缩型标志物α-平滑肌肌动蛋白(α-SMA)、合成型标志物骨桥蛋白(OPN)及CRY1 mRNA和蛋白的表达;分别构建CRY1的干扰RNA(siCRY1)、YAP1的干扰RNA(siYAP1)进行基因沉默,构建CRY1的过表达质粒(pcDNA-CRY1)进行过表达,RT-PCR、Western blot双重验证各基因组mRNA和蛋白的表达;将VSMCs分为4组,对照组、AngⅡ组、AngⅡ+siCRY1组、AngⅡ+pcDNA-CRY1组,将siCRY1、siYAP1、pcDNA-CRY1转染至经AngⅡ处理VSMCs中,RT-PCR和Western blot检测α-SMA、OPN、CRY1、YAP1 mRNA和蛋白的表达及CCK-8法检测细胞增殖情况。结果不同浓度AngⅡ刺激VSMCs不同时间后,VSMCs收缩型标志物表达减少,VSMCs合成型标志物表达增加,CRY1表达减少;转染siCRY1、siYAP1、pcDNA-CRY1处理VSMCs后,CRY1在siCRY1组中表达下降,在pcDNA-CRY1组中表达升高,YAP1在siYAP1组中表达下降,在siCRY1组中表达升高;转染siCRY1进一步减少AngⅡ诱导VSMC收缩型标志物的表达(P<0.05),增加合成型标志物、YAP1的表达,以及促进VSMCs的增殖(P<0.05);转染pcDNA-CRY1质粒增加AngⅡ诱导VSMCs收缩型标志物的表达(P<0.05),减弱合成型标志物YAP1的表达(P<0.05)及抑制VSMCs的增殖(P<0.05)。结论生物钟蛋白CRY1抑制AngⅡ诱导的小鼠主动脉VSMCs表型蛋白标志物变化和增殖,该过程可能与Hippo-YAP信号通路有关。Objective To investigate the effect of circadian clock protein cryptochrome 1(CRY1)on angiotensinⅡ(AngⅡ)-induced phenotypic transformation of mouse aorta smooth muscular cells.Methods VSMCs were divided into control group and experimental group.VSMCs were treated with different concentrations of AngⅡfor various time,α-smooth muscle actin(α-SMA),osteopontin(OPN)and CRY1 expressions in VSMCs were detected via RT-qPCR and Western blot.VSMCs were transfected with knockdown vector siCRY1 and siYAP1 or overexpression vector,and the corresponding gene expression was detected with RT-PCR and Western blot.VSMCs were divided into 4 groups:Control group,AngⅡgroup,AngⅡ+siCRY1 group and AngⅡ+pcDNA-CRY1 group.siCRY1,siYAP1,pcDNA-CRY1 were transfected into VSMCs induced by AngⅡfor observation onα-SMA,CRY1,OPN and YAP1 at mRNA and protein levels detected by RT-PCR and Western blot.CCK-8 method were used to measure proliferation capacity.Results VSMCs were treated with different concentrations of AngⅡfor various time,the expression of CRY1 was decreased in a model of AngⅡ-induced VSMC phenotypic transformation,which contractile markers down-regulated and synthetic markers up-regulated at mRNA and protein levels.After VSMCs were transfected with siCRY1,siYAP1 and pcDNA-CRY1,the expression of CRY1 decreased insiCRY1 group and increased in pcDNA-CRY1 group,while the expression of YAP1 decreased insiYAP1 group and increased insiCRY1 group.Knockdown of CRY1 could stimulate the transformation of VSMCs from contractile phenotype into synthetic phenotypes by further activating expression ofsynthetic markers and CRY1(P<0.05),inhibiting expression of contractile markers and YAP1(P<0.05),the proliferation of VSMCs increased significantly(P<0.05).While overexpressed CRY1 could block the phenotypic transformation,the expression ofsynthetic markers and CRY1 was reduced and contractile markers and YAP1 was increased both at mRNA and protein levels(P<0.05),the proliferation of VSMCs inhibited(P<0.05).Conclusions CRY1 may inh

关 键 词：隐花色素1 Yes相关蛋白1 血管紧张素Ⅱ 表型转化 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学] R363[医药卫生—基础医学]