细胞外基质刚度通过FAK/ROCK-1/YAP通路调控尿道成纤维细胞活化  

Extracellular matrix stiffness regulates the activation of urethral fibroblasts via FAK/ROCK-1/YAP pathway

作　　者：陈烨辉 陈佳茵 朱君明 陈航 柯志滨 林菲 薛学义[1] 魏勇[1] 郑清水[1] 许宁[1] Chen Yehui;Chen Jiayin;Zhu Junming;Chen Hang;Ke Zhibin;Lin Fei;Xue Xueyi;Wei Yong;Zheng Qingshui;Xu Ning(Department of Urology,the First Affiliated Hospital of Fujian Medical University,Fuzhou 350005,China)

机构地区：[1]福建医科大学附属第一医院泌尿外科,福州350005

出　　处：《中华实验外科杂志》2022年第3期500-502,共3页Chinese Journal of Experimental Surgery

基　　金：福建省财政专项(2019B030);福建省自然科学基金(2020J01989)。

摘　　要：目的探讨基质刚度对尿道成纤维细胞(UFBs)活化的影响及其机制。方法2020年6月至2021年2月从福建医科大学附属第一医院泌尿外科手术患者获得的尿道瘢痕组织中提取原代UFBs;用聚二甲基矽氧烷分别以60∶1(Soft)、40∶1(Moderate)、30∶1(Stiff)的比例加入固化剂,制备不同刚度的基质凝胶,将UFBs接种其上培养。倒置显微镜观察在不同刚度基质凝胶上生长的UFBs形态学特点;蛋白质印迹法(Western blot)检测UFBs活化标志物α-平滑肌肌动蛋白(α-SMA)、Ⅰ型胶原蛋白(collagenⅠ)和Ⅲ型胶原蛋白(collagenⅢ)表达水平;进一步检测FAK/ROCK-1/YAP通路标志蛋白表达水平,并观察整合素抑制剂Cilengtide(20 ng/ml)对其影响,探索基质刚度是否通过此通路影响UFBs活化;采用方差分析和独立样本t检验比较组间差异。结果随着基质凝胶刚度的增加,接种其上的UFBs形态由椭圆形转变为偏梭形,细胞伪足增长、增多;Soft、Moderate、Stiff组UFBs的α-SMA相对表达量分别为(0.140±0.004、0.815±0.030和1.385±0.051,F=989.247,P<0.05);collagenⅠ相对表达量分别为(0.138±0.004、0.947±0.021和1.263±0.028,F=2415.159,P<0.05);collagenⅢ相对表达量分别为(0.284±0.005、1.089±0.017和1.374±0.038,F=1661.310,P<0.05),均随基质刚度增加而升高;Stiff组中UFBs的p-FAK(1.282±0.029比0.430±0.009,t=48.262,P<0.05)、ROCK-1(1.366±0.111比0.474±0.034,t=13.300,P<0.05)和YAP(1.272±0.020比0.908±0.007,t=30.254,P<0.05)表达水平均显著高于Soft组,而p-YAP(0.589±0.006比1.028±0.008,t=-74.860,P<0.05)表达水平显著低于Soft组;在加入整合素抑制剂Cilengtide后,基质刚度增加引起的p-FAK、ROCK-1、YAP和p-YAP表达改变均减弱。结论细胞外基质刚度通过整合素介导的FAK/ROCK-1/YAP通路调控人尿道成纤维细胞活化。Objective To investigate the effects and mechanism of extracellular matrix stiffness on the activation of urethral fibroblasts(UFBs).Methods UFBs extracted from urethral scar tissues derived from patients who underwent surgery in Department of Urology,the First Affiliated Hospital of Fujian Medical,between June 2020 and February 2021,were seeded on the prepared matrigels of different matrix stiffness,which were made using polydimethylsiloxane with hardener(60∶1 for soft group,40∶1 for moderate group,and 30∶1 for stiff group),and cultured for follow-up experiments.Morphologic characteristics of UFBs cultured on matrigels of different matrix stiffness were observed.Western blotting was performed to detect the expression levels ofα-smooth muscle actin(α-SMA),collagenⅠand collagenⅢ.The expression levels of FAK/ROCK-1/YAP pathway markers,and the effects of integrin inhibitor Cilengtide(20 ng/ml)on the pathway were also examined,to investigate whether extracellular matrix stiffness regulated the activation of UFBs via this signaling pathway.ANOVA and Student t test were used for statistical analysis.Results The shape of UFBs changed from elliptic to fusiform and the pseudopodias of UFBs became more and longer,with the matrix stiffness of matrigel increasing.The relative expression levels ofα-SMA,collagenⅠand collagenⅢin the soft,moderate and stiff groups were 0.140±0.004,0.815±0.030 and 1.385±0.051(F=989.247,P<0.05),(0.138±0.004,0.947±0.021 and 1.263±0.028(F=2415.159,P<0.05),and 0.138±0.004,0.947±0.021 and 1.263±0.028(F=1661.310,P<0.05),respectively,which indicated that the expression levels ofα-SMA,collagenⅠand collagenⅢwere elevated along with the increase of extracellular matrix stiffness.When compared with the soft group,the expression levels of p-FAK(1.282±0.029 vs.0.430±0.009,t=48.262,P<0.05),ROCK-1(1.366±0.111 vs.0.474±0.034,t=13.300,P<0.05)and YAP(1.272±0.020 vs.0.908±0.007,t=30.254,P<0.05)in the stiff group were increased,while the expression level of p-YAP(0.589±0.006 vs.

关 键 词：尿道成纤维细胞 FAK/ROCK-1/YAP通路 尿道狭窄 

分 类 号：R695[医药卫生—泌尿科学]