猪脱细胞真皮基质通过激活Wnt/β-连环蛋白信号通路对大鼠角质形成细胞细胞周期蛋白D1的表达的影响  

Effect of porcine acellular dermal matrix on Cyclin D1 expression in rat keratinocytes by activating Wnt/β-catenin signal pathway

作　　者：陈冉 李恭驰[2] 李炳辉[1] 邓海波 张晨晨 邝莉雯 杜烨 周云华 郑洁[1] Chen Ran;Li Gongchi;Li Binghui;Deng Haibo;Zhang Chenchen;Kuang Liwen;Du Ye;Zhou Yunhua;Zheng Jie(Department of Wound Repair,Liyuan Hospital Affiliated to Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430077,China;Department of Hand Surgery,Union Hospital,Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430022,China)

机构地区：[1]华中科技大学同济医学院附属梨园医院创面修复科,武汉430077 [2]华中科技大学同济医学院附属协和医院手外科,武汉430022

出　　处：《中华实验外科杂志》2021年第8期1475-1478,共4页Chinese Journal of Experimental Surgery

基　　金：国家重大疾病多学科合作诊疗能力建设项目(国卫办医函2019542);国家自然科学基金青年科学基金项目(81801922);2020湖北省自然科学基金基金项目(2020CFB696);2020湖北省重点研发计划项目(2020BCB029);湖北省慢性创面及糖尿病医学临床研究中心资助项目(2018BCC340)。

摘　　要：目的探讨猪脱细胞真皮基质(pADM)对大鼠角质形成细胞细胞周期蛋白(Cyclin)D1的影响及其机制。方法体外培养原代大鼠角质形成细胞,免疫组织化学鉴定原代角质形成细胞;实验分组:对照组、pADM组、Wnt/β-连环蛋白(β-catenin)信号通路激动剂SKL2001组、pADM+Wnt/β-catenin信号通路抑制剂FH535组;采用荧光定量聚合酶链反应(qPCR)和蛋白质印迹法(Western blot)分析大鼠角质形成细胞中Wnt/β-catenin信号通路相关分子以及细胞周期蛋白Cyclin D1的表达。两组间比较采用非配对t检验,多组间比较采用单因素方差分析。结果pADM组Wnt/β-catenin信号通路相关分子Wnt3a,β-catenin,LEF-1和细胞周期蛋白Cyclin D1表达水平高于对照组(Wnt3a,qPCR:2.489±0.414比1.210±0.242,t=4.068,P<0.05;Western blot:0.571±0.043比0.217±0.071,t=7.401,P<0.05;β-catenin,qPCR:2.594±0.244比0.818±0.188,t=9.877,P<0.05;Western blot:0.794±0.031比0.420±0.026,t=16.12,P<0.05;LEF-1,qPCR:2.816±0.190比0.896±0.090,t=9.702,P<0.05;Western blot:0.700±0.124比0.276±0.090,t=4.781,P<0.05;Cyclin D1,qPCR:2.708±0.305比0.922±0.095,t=15.720,P<0.05;Western blot:0.554±0.097比0.166±0.067,t=5.723,P<0.05);pADM+FH535组Wnt3a,β-catenin,LEF-1和Cyclin D1表达水平低于pADM组(Wnt3a,qPCR:0.350±0.049比0.964±0.159,F=49.890,P<0.05;Western blot:0.192±0.106比0.485±0.028,F=47.780,P<0.05;β-catenin,qPCR:0.431±0.149比0.919±0.074,F=39.430,P<0.05;Western blot:0.174±0.013比0.386±0.048,F=49.950,P<0.05;LEF-1,qPCR:0.502±0.067比0.954±0.112,F=79.130,P<0.05;Western blot:0.091±0.037比0.377±0.055,F=121.000,P<0.05;Cyclin D1,qPCR:0.264±0.049比0.962±0.037,F=105.500,P<0.05;Western blot:0.111±0.053比0.297±0.023,F=137.300,P<0.05)。结论pADM有类似于Wnt/β-catenin信号通路激动剂的作用,其可通过激活Wnt/β-catenin信号通路提高大鼠角质形成细胞中Cyclin D1的表达,FH535可阻断pADM对Cyclin D1的激活作用。Objective To investigate the effect of porcine acellular dermal matrix(pADM)on Cyclin D1 of rat keratinocytes and its mechanism.Methods Primary rat keratinocytes were cultured in vitro and identified by immunohistochemistry.Following experimental groups were established:control group,pADM group,Wnt/β-catenin signal pathway agonist SKL2001 group,pADM+Wnt/β-catenin signal pathway inhibitor FH535 group.The fluorescence quantitative PCR(qPCR)and the Western blotting were used to analyze the expression of Wnt/β-catenin signal pathway related molecules and Cyclin D1 in rat keratinocytes.Data were analyzed by Graph Pad Prism 8,and the measurement data were expressed by mean±standard deviation(x±s).Unpaired t-test was used for comparison between the two groups,and one-way ANOVA was used for comparison between multiple groups.Results The expression levels of Wnt/β-catenin signal pathway related molecules Wnt3a,β-catenin,LEF-1 and Cyclin D1 in pADM group were significantly higher than those in control group(for Wnt3a,qPCR:2.489±0.414 vs.1.210±0.242,t=4.068,P<0.05;Western blotting:0.571±0.043 vs.0.217±0.071,t=7.401,P<0.05;for β-catenin,qPCR:2.594±0.244 vs.0.818±0.188,t=9.877,P<0.05;Western blotting:0.794±0.031 vs.0.420±0.026,t=16.12,P<0.05);for(LEF-1,qPCR:2.816±0.190 vs.0.896±0.090,t=9.702,P<0.05;Western blotting:0.700±0.124 vs.0.276±0.090,t=4.781,P<0.05;for Cyclin D1,qPCR:2.708±0.305 vs.0.922±0.095,t=15.720,P<0.05;Western blotting:0.554±0.097 vs.0.166±0.067,t=5.723,P<0.05).The expression levels of Wnt3a,β-catenin,LEF-1 and Cyclin D1 in pADM+FH535 group were significantly lower than those in pADM group(for Wnt3a,qPCR:0.350±0.049 vs.0.964±0.159,F=49.890,P<0.05;Western blotting:0.192±0.106 vs.0.485±0.028,F=47.780,P<0.05;forβ-catenin,qPCR:0.431±0.149 vs.0.919±0.074,F=39.430,P<0.05;Western blotting:0.174±0.013 vs.0.386±0.048,F=49.950,P<0.05;for LEF-1,qPCR:0.502±0.067 vs.0.954±0.112,F=79.130,P<0.05;Western blotting:0.091±0.037 vs.0.377±0.055,F=121.000,P<0.05;for Cyclin D1,qPCR:0.264±0.049 v

关 键 词：猪脱细胞真皮基质 WNT信号通路 角质形成细胞 细胞周期 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]