整合素αV在应力刺激促心脏成纤维细胞活化中的作用机制研究  

Role of integrinαV in mediating cardiac fibroblasts activation induced by mechanical stress

作　　者：程培培 丰明会 李赞 陈誉文 吕嵘[1] 徐明 CHENG Pei-pei;FENG Ming-hui;LI Zan;CHEN Yu-wen;LU Rong;XU Ming(School of Basic Medicine,Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China;Jiading Branch of Shanghai General Hospital,Shanghai 201803,China)

机构地区：[1]上海中医药大学基础医学院,上海201203 [2]上海市第一人民医院嘉定分院,上海201803

出　　处：《中国病理生理杂志》2022年第8期1345-1353,共9页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.81873033);上海市进一步加快中医药事业发展三年行动计划项目(2018年-2020年)[No.ZY(2018-2020)-CCCX-2001-01]。

摘　　要：目的:探究应力刺激对心脏成纤维细胞活化的影响及整合素αV(integrin αV)在其中可能的作用及其机制。方法:原代1周龄C57BL/6小鼠心脏成纤维细胞,分别以鼠尾I型胶原蛋白(collagen type Ⅰ,Col Ⅰ)制备胶和聚苯乙烯材质为软、硬基质培养条件,并结合siRNA下调硬基质培养的心脏成纤维细胞的integrin αV表达,将细胞分为软基质组(soft组)、硬基质组(stiff组)、siRNA阴性对照组(NC组)和integrin αV siRNA组(siRNA组)。显微镜下白场成像评价心脏成纤维细胞形态及其伪足数量和形态;CCK-8及免疫荧光法检测心脏成纤维细胞活力和增殖能力;划痕实验检测心脏成纤维细胞迁移能力;Western blot法检测Col Ⅰ、α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、Ⅲ型胶原蛋白(collagen type Ⅲ,Col Ⅲ)、integrin αV、黏着斑激酶(focal adhesion kinase,FAK)、pFAK、蛋白激酶B(protein kinase B,PKB/Akt)及p-Akt的蛋白水平。结果:与soft组相比,stiff组心脏成纤维细胞表面积显著增大,伪足由丝状向板状演变(P<0.01),增殖能力及合成和分泌Col Ⅰ和Col Ⅲ能力均显著增加(P<0.01),α-SMA蛋白表达增加(P<0.01),向肌成纤维细胞表型转化,同时integrin αV/FAK/Akt信号通路被激活。下调stiff组心脏成纤维细胞的integrin αV表达后,细胞板状伪足回缩,迁移能力降低(P<0.05),合成和分泌Col Ⅰ和Col Ⅲ能力降低(P<0.01),同时抑制integrin αV/FAK/Akt信号通路的激活(P<0.01)。结论:应力刺激通过integrin αV介导的FAK/Akt信号通路激活促使心脏成纤维细胞转分化为更活跃的肌成纤维细胞,发挥抗应力和细胞外基质蛋白重组的作用,从而参与心脏疾病的病理转归。AIM:To explore the effect of stress stimulation on cardiac fibroblasts(CFs)and the possible mechanism of integrin αV involved in this process.METHODS:Primary CFs isolated from one-week-old C57BL/6 mice were cultured in rat tail collagen type Ⅰ(Col Ⅰ)gel and polystyrene dishes as soft and stiff matrix culture conditions,respectively. Small interfering RNA(siRNA)was used to silence integrin αV in the CFs cultured in stiff condition. The cells were divided into soft matrix group(soft group),stiff matrix group(stiff group),siRNA negative control(NC)group(NC group),and integrin αV siRNA group(siRNA group). The morphological changes of the cells and the number of pseudopods were evaluated by bright-field microscopy. Cell viability and proliferation were assessed by CCK-8 and Ki67immunofluorescence staining. Cell migration ability was measured by scratch test. Western blot was applied to detect the protein levels of α-smooth muscle actin(α-SMA),Col Ⅰ,collagen type Ⅲ(Col Ⅲ),integrin αV,focal adhesion kinase(FAK),p-PAK,protein kinase B(PKB/Akt)and p-Akt.RESULTS:Compared with the CFs cultured in soft matrix,the surface area of thr CFs cultured in stiff matrix increased significantly,and the cells exhibited filopodia to lamellipodia transition(P<0. 01),with enhanced cell proliferation and increased synthesis of α-SMA,Col Ⅰ and Col Ⅲ(P<0. 01),indicating a phenotypic switch of quiescent CFs to activated myofibroblasts(MFBs). Meanwhile,integrin αV and its downstream FAK/Akt pathway were activated in stiff group(P<0. 01). However,silencing of integrin αV in the CFs cultured in stiff matrix resulted in retraction of lamellipodia,reduced cell mobility(P<0. 05),partially blunted Col Ⅰ and Col Ⅲ production(P<0. 01)and FAK-Akt signal(P<0. 01).CONCLUSION:The MFBs transdifferentiates from quiescent CFs upon mechanical stress via integrin αV-activated integrin αV/FAK/Akt signaling pathway,which participates in the pathological remodeling of diseased heart due to their anti-stress effect and extracellular ma

关 键 词：心脏成纤维细胞 应力刺激 整合素ΑV 黏着斑激酶 蛋白激酶B 

分 类 号：R542.2[医药卫生—心血管疾病] R363.1[医药卫生—内科学]