钙联蛋白在心肌成纤维细胞活化过程中的作用研究  

The potential role of calnexin in the activation of cardiac fibroblasts

作　　者：田格尔 赵明月[1] 周骏腾 权月 吴文超[1] 刘小菁[1,2] TIAN Geer;ZHAO Mingyue;ZHOU Junteng;QUAN Yue;WU Wenchao;LIU Xiaojing(Laboratory of Cardiovascular Diseases,Regenerative Medicine Research Center,West China Hospital,Sichuan University,Chengdu 610041,P.R.China;Department of Cardiology,West China Hospital,Sichuan University,Chengdu 610041,P.R.China)

机构地区：[1]四川大学华西医院再生医学研究中心心血管疾病研究室,成都610041 [2]四川大学华西医院心内科,成都610041

出　　处：《生物医学工程学杂志》2020年第3期450-459,共10页Journal of Biomedical Engineering

基　　金：国家自然科学基金(11672197)。

摘　　要：钙联蛋白(Calnexin)是内质网上的凝集素样分子伴侣蛋白,也是线粒体结合内质网膜上的重要蛋白,在心脏疾病中发挥着重要作用,但其在心肌成纤维细胞活化中的作用和机制并未阐明。本研究通过小鼠胸主动脉缩窄(TAC)模型来观察体内心肌纤维化情况,并利用心脏超声,苏木精-伊红、Masson和天狼星红染色,以及心脏纤维化标志物表达水平,鉴定模型是否成功。体外实验利用转化生长因子TGFβ1刺激建立心肌成纤维细胞活化模型,通过定量PCR、Western blot、EdU渗入法及划痕实验等方法检测其活化、细胞增殖、迁移的变化。利用腺病毒过表达和沉默Calnexin,来观察Calnexin在体外心肌成纤维细胞活化中的作用。结果显示,Calnexin在TAC模型和体外心肌成纤维细胞活化过程中均表达降低。过表达Calnexin能缓解心肌成纤维细胞活化;相反,沉默Calnexin导致其活化加重。进一步探究发现,内质网应激在心肌成纤维细胞活化过程中被激活,过表达Calnexin后内质网应激缓解,相反,敲低Calnexin后内质网应激加重,与活化表型变化一致。结果表明,过表达Calnexin可能通过缓解内质网应激从而减轻心肌成纤维细胞的活化程度。Calnexin is a lectin-like molecular chaperone protein on the endoplasmic reticulum,mediating unfolded protein responses,the endoplasmic reticulum Ca2 homeostasis,and Ca2 signals conduction.In recent years,studies have found that calnexin plays a key role in the heart diseases.This study aims to explore the role of calnexin in the activation of cardiac fibroblasts.A transverse aortic constriction(TAC)mouse model was established to observe the activation of cardiac fibroblasts in vivo,and the in vitro cardiac fibroblasts activation model was established by transforming growth factorβ1(TGFβ1)stimulation.The adenovirus was respectively used to gene overexpression and silencing calnexin in cardiac fibroblasts to elucidate the relationship between calnexin and cardiac fibroblasts activation,as well as the possible underlying mechanism.We confirmed the establishment of TAC model by echocardiography,hematoxylin-eosin,Masson,and Sirius red staining,and detecting the expression of cardiac fibrosis markers in cardiac tissues.After TGFβ1 stimulation,markers of the activation of cardiac fibroblast,and proliferation and migration of cardiac fibroblast were detected by quantitative PCR,Western blot,EdU assay,and wound healing assay respectively.The results showed that the calnexin expression was reduced in both the TAC mice model and the activated cardiac fibroblasts.The overexpression of calnexin relieved cardiac fibroblasts activation,in contrast,the silencing of calnexin promoted cardiac fibroblasts activation.Furthermore,we found that the endoplasmic reticulum stress was activated during cardiac fibroblasts activation,and endoplasmic reticulum stress was relieved after overexpression of calnexin.Conversely,after the silencing of calnexin,endoplasmic reticulum stress was further aggravated,accompanying with the activation of cardiac fibroblasts.Our data suggest that the overexpression of calnexin may prevent cardiac fibroblasts against activation by alleviating endoplasmic reticulum stress.

关 键 词：钙联蛋白 胸主动脉缩窄 心肌纤维化 心肌成纤维细胞活化 内质网应激 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]