1、中国病理生理杂志 Chinese Journal of Pathophysiology 2023,39(2):204-211杂志网址:http:/CIRBP对小鼠血管平滑肌细胞增殖及迁移的调控及机制研究*赵嘉琪1,杨大春2,王强2,孙雄山2,杨永健1,2(1西南医科大学临床医学院,四川 泸州646000;2西部战区总医院心内科,四川 成都 610083)摘要 目的:探讨冷诱导RNA结合蛋白(CIRBP)对小鼠血管平滑肌细胞(VSMCs)增殖及迁移过程的调控及机制。方法:将雄性C57BL/6J小鼠分为假手术对照组、假手术干扰组、颈总动脉损伤对照组和颈总动脉损伤干扰组,每组5只。造模后按照组别分别
2、用阴性对照腺病毒(AD-NC)和沉默CIRBP腺病毒(AD-CIRBPI)转染,28 d后观察 CIRBP表达及血管内膜的增生情况。将小鼠 VSMCs分为对照组和腺病毒沉默组,分别用 AD-NC和 AD-CIRBPI 转染细胞 48 h,然后加入激活雷帕霉素靶蛋白复合物 1(mTORC1)活性的胰岛素。通过 RT-qPCR 检测CIRBP 的 mRNA 表达,Western blot 检测 CIRBP、磷酸化核糖体蛋白 S6(p-S6Ser235/236)和磷酸化 4E 结合蛋白 1(p-4EBP1Thr37/46)蛋白水平变化,Ki67免疫荧光染色和CCK-8实验检测细胞增殖,划痕实验检测细
3、胞迁移,HE染色检测颈动脉内膜增生程度。结果:沉默CIRBP后,VSMCs的活力下降,Ki67阳性细胞比率降低,细胞迁移速度减慢,同时mTORC1活性下降。加入胰岛素恢复mTORC1活性后,细胞活力、Ki67阳性细胞率和细胞迁移速度下降幅度减弱。损伤小鼠颈总动脉内皮后CIRBP表达增加,体内干扰小鼠CIRBP表达后,小鼠血管内皮损伤后内膜增生程度减轻。结论:CIRBP通过mTORC1途径加强小鼠VSMCs增殖及迁移,促进小鼠血管损伤后血管内膜增生。关键词 血管平滑肌细胞;细胞增殖;细胞迁移;冷诱导RNA结合蛋白;哺乳动物雷帕霉素靶蛋白复合物1中图分类号 R543.5;R363.2+1 文献标志
4、码 A doi:10.3969/j.issn.1000-4718.2023.02.002Regulatory effect of cold-inducible RNA-binding protein on proliferation and migration of mouse vascular smooth muscle cells and its mechanismZHAO Jiaqi1,YANG Dachun2,WANG Qiang2,SUN Xiongshan2,YANG Yongjian1,2(1School of Clinical Medicine,Southwest Medica
5、l University,Luzhou 646000,China;2Department of Cardiovascular Medicine,The General Hospital of Western Theater Command,Chengdu 610083,China.E-mail:;)ABSTRACT AIM:To investigate the regulatory effect of cold-inducible RNA binding protein(CIRBP)on the proliferation and migration of mouse vascular smo
6、oth muscle cells(VSMCs),and to explore its mechanism.METHODS:Male C57BL/6J mice were divided into 4 groups:sham+adenovirus-negative control(AD-NC)group,sham+adenovirus-CIRBP inhibitor(AD-CIRBPI)group,carotid vascular injury+AD-NC group,and carotid vascular injury+AD-CIRBPI group,with 5 mice in each
7、group.After the injury model was established,the mice were transfected with AD-NC or AD-CIRBPI.The CIRBP expression and vascular intimal hyperplasia were measured at 28 d after injury.Mouse VSMCs were divided into AD-NC and AD-CIRBPI group.The cells of these two groups were transfected with AD-NC an
8、d AD-CIRBPI for 48 h,respectively.The activity of mammalian target of rapamycin complex 1(mTORC1)was restored by insulin.The mRNA expression of CIRBP was explored by RT-qPCR.The levels of CIRBP,phosphorylated ribosomal protein S6(p-S6Ser235/236)and phosphorylated 4E-binding protein 1(p-4EBP1Thr37/46
9、)were detected by Western blot.The cell proliferation was explored by Ki67 immunofluorescence staining and CCK-8 assay.The cell migration was determined by scratch test.The carotid intimal hyperplasia was determined by HE staining.RESULTS:The viability of VSMCs,the ratio of Ki67-positive cells,wound
10、-healing rate and the activity of mTORC1 were decreased after silencing of CIRBP.However,the inhibitory effects of CIRBP silencing on cell viability,ratio of Ki67-positive cells and wound-healing rate were attenuated af文章编号 1000-4718(2023)02-0204-08 收稿日期 2022-09-16 修回日期 2022-11-04*基金项目 国家自然科学基金资助项目(
11、No.82070289);四川省自然科学基金资助项目(No.2022NSFSC0820)通讯作者 杨永健 Tel:028-86571250;E-mail:;孙雄山 Tel:028-86571255;E-mail:204ter restoring the activity of mTORC1 by insulin.The expression of CIRBP was increased after mouse common carotid vascular injury.The vascular intimal hyperplasia was attenuated after interfer
12、ing with the expression of CIRBP in vivo.CONCLUSION:CIRBP promotes the proliferation and migration of mouse VSMCs,and vascular intimal hyperplasia after mouse common carotid vascular injury through the mTORC1 pathway.KEY WORDS vascular smooth muscle cells;cell proliferation;cell migration;cold-induc
13、ible RNA-binding protein;mammalian target of rapamycin complex 1在我国,患心血管病的人数在迅速增加,其中动脉粥样硬化性心血管病的患病率与死亡率逐年上升,并且有年轻化的趋势1-2。经皮冠脉介入治疗(percutaneous coronary intervention,PCI)是主要解决方法,但术后血管再狭窄严重制约PCI的预后。血管内皮细胞受到机械损伤后,经过内皮剥落,血小板活化,促炎细胞因子和趋化因子的释放以及血管平滑肌细胞移动到内皮下等过程,最终使血管内膜增生,引起血管内再狭窄3。当前血管平滑肌细胞增殖的分子调控机制主要包括Ra
14、s/丝裂原活化蛋白激酶、Janus激酶/信号转导及转录激活因子等4,针对血管内再狭窄的治疗手段进步显著,但术后血管再狭窄的发生率仍有5%10%5,提示仍有未知的分子机制需要探索。冷 诱 导 RNA 结 合 蛋 白(cold-inducible RNA-binding protein,CIRBP)是一种冷休克相关蛋白,属于RNA结合蛋白家族,在冷应激等条件下可以促进翻译6。CIRBP能调控器官与组织细胞的生长、增殖和分化7、维持端粒酶活性8、抑制细胞凋亡9、促进炎症细胞因子的激活与释放10。多个研究表明CIRBP在心力衰竭和血管损伤等心血管疾病中有重要意义10-14,当血管受到各种损伤和病理因素
15、刺激时,血管平滑肌细胞会从分化状态转变为去分化状态,增殖异常活跃,并进一步向内膜迁移,导致内膜增生及参与动脉粥样硬化、肺动脉高压等心血管疾病的进展15。既往研究显示CIRBP在乳腺癌、黑色素瘤、垂体腺瘤和胰腺导管腺癌等肿瘤细胞中发挥促进肿瘤细胞增殖作用16-19。但是CIRBP对血管平滑肌细胞增殖、迁移和血管内膜增生引起血管内再狭窄的调控目前并不清楚。本研究通过机械损伤小鼠颈总动脉内皮的方法构建血管再狭窄模型,用腺病毒感染方法干扰CIRBP表达,探究CIRBP在小鼠VSMCs增殖、迁移和血管内膜增生中的作用机制,为PCI术后防治血管再狭窄提供参考资料。材料和方法1动物20只约 7周龄 SPF级
16、的雄性 C57BL/6J小鼠,重约25g(成都药康生物科技有限公司),许可证编号:SCXK(川)2020-034。小鼠先适应性培养1周,白天适当光照12 h,恒温通风,喂以水和食物。2细胞和主要试剂小鼠主动脉平滑肌细胞系MOVAS(深圳市豪地华拓生物科技有限公司);RT-qPCR实验试剂盒(TaKaRa);RT-qPCR实验所用的引物(成都擎科伟业生物技术有限公司);DMEM高糖培养液、0.25%胰蛋白酶及青霉素+链霉素双抗(HyClone);胎牛血清(Gibco);磷酸盐缓冲液(phosphate-buffered saline,PBS)、5%牛 血 清 白 蛋 白(bovine serum albumin,BSA)和CIRBP抗体(武汉三鹰生物科技有限公司);4E结合蛋白1(4E-binding protein 1,4EBP1)抗体、磷酸化4EBP1(phosphorylated 4EBP1,p-4EBP1Thr37/46)抗体、GAPDH 单克隆抗体、核糖体蛋白 S6(ribosomal protein S6,S6)抗体、磷酸化S6(phosphorylated S6,p-S6S