ROCK1基因对瘢痕疙瘩成纤维细胞增殖与迁移及相关分子表达的影响  

Effects of the ROCK1 gene on proliferation and migration of and related molecular expression in keloid fibroblasts

作　　者：桑鹏飞 方明松[1] 李旋[1] 曹林[1] 赵玲玲 刘畅[1] 蒋智永 朱飞[2] Sang Pengfei;Fang Mingsong;Li Xuan;Cao Lin;Zhao Lingling;Liu Chang;Jiang Zhiyong;Zhu Fei(Department of Plastic Surgery,The Second People′s Hospital of Hefei(Hefei Hospital Affiliated to Anhui Medical University),Hefei 230041,China;Department of Plastic Surgery,The First Affiliated Hospital of Anhui Medical University,Hefei 230022,China)

机构地区：[1]合肥市第二人民医院安徽医科大学附属合肥医院整形外科,合肥230041 [2]安徽医科大学第一附属医院整形外科,合肥230022

出　　处：《中华皮肤科杂志》2023年第3期222-228,共7页Chinese Journal of Dermatology

基　　金：安徽高校省级自然科学研究重点项目(KJ2014A108);安徽医科大学校科研基金(2019xkj094)。

摘　　要：目的研究ROCK1基因对瘢痕疙瘩成纤维细胞增殖和迁移能力及相关分子表达的影响。方法免疫组化检测人瘢痕疙瘩和正常皮肤组织中ROCK1蛋白的表达,Western印迹检测人瘢痕疙瘩组织中ROCK1、转化生长因子β1(TGF-β1)和E钙黏附蛋白(E-Cad)的表达。体外培养人瘢痕疙瘩成纤维细胞(HKF),分为ROCK1基因过表达对照组(ROCK1 NC组,转染ROCK1基因过表达对照载体)、ROCK1基因过表达组(ROCK1 OE组,转染ROCK1基因过表达载体)、ROCK1基因干扰对照组(sh NC组,转染ROCK1基因干扰对照载体)、ROCK1基因干扰组(shROCK1组,转染ROCK1基因干扰载体)共4组。细胞计数试剂盒8(CCK8)检测ROCK1基因对HKF存活率的影响;Transwell小室检测ROCK1基因对HKF迁移的影响;实时荧光定量PCR、免疫印迹检测ROCK1、TGF-β1和E-Cad基因mRNA和蛋白的表达。结果免疫组化检测显示,人瘢痕疙瘩组织中ROCK1表达较正常组织显著降低(t=6.47,P=0.003);Western印迹检测显示,与正常组织相比,人瘢痕疙瘩组织中ROCK1、E-Cad蛋白表达显著降低(t值分别为14.02、162.20,均P<0.001),TGF-β1蛋白表达显著升高(t=76.01,P<0.001)。CCK8测定显示,转染24 h后,ROCK1 OE组细胞活性显著低于ROCK1 NC组(t值分别为3.25、3.78,P值分别为0.031、0.019),shROCK1组细胞活性显著高于sh NC组(t值分别为3.12、2.79,P值分别为0.036、0.049)。ROCK1 OE组迁移细胞数显著低于ROCK1 NC组(t=5.17,P=0.004),shROCK1组迁移细胞数显著高于sh NC组(t=9.28,P<0.001)。ROCK1 OE组与ROCK1 NC组相比,ROCK1、E-Cad mRNA和蛋白表达量显著升高(P<0.05或<0.001),TGF-β1 mRNA和蛋白表达量显著降低(均P<0.001);shROCK1组与sh NC组相比,ROCK1、E-Cad mRNA和蛋白表达量显著降低(P<0.05或<0.001),TGF-β1 mRNA和蛋白表达量显著升高(P=0.005或<0.001)。结论ROCK1基因能抑制HKF的增殖及迁移,过表达ROCK1基因能下调TGF-β1基因表达,上调E-Cad基因表达。Objective To investigate effects of the ROCK1 gene on proliferation and migration of and related molecular expression in keloid fibroblasts.Methods Immunohistochemical technique was used to detect ROCK1 protein expression in human keloids and normal skin tissues,and Western blot analysis was performed to detect the expression of ROCK1,transforming growth factorβ1(TGF-β1)and E-cadherin in keloid tissues.In vitro cultured human keloid fibroblasts(HKFs)were divided into 4 groups:ROCK1 gene overexpression control group(ROCK1 NC group)transfected with ROCK1 gene overexpression control vectors,ROCK1 gene overexpression group(ROCK1 OE group)transfected with ROCK1 gene overexpression vectors,ROCK1 gene knockdown control group(sh NC group)transfected with ROCK1 gene knockdown control vectors,and ROCK1 gene knockdown group(shROCK1 group)transfected with ROCK1 gene knockdown vectors.Cell counting kit-8(CCK8)assay was performed to evaluate the effect of ROCK1 gene on the survival rate of HKFs,Transwell assay to evaluate the effect on the migration of HKFs,and real-time fluorescence-based quantitative PCR(qRT-PCR)and Western blot analysis were conducted to determine the mRNA and protein expression of ROCK1,TGF-β1 and E-cadherin,respectively.Results Immunohistochemical study showed that ROCK1 protein expression decreased significantly in the human keloid tissues compared with the normal tissues(t=6.47,P=0.003);Western blot analysis showed that the expression levels of ROCK1 and E-cadherin significantly decreased(t=14.02,162.20,respectively,both P<0.001),while TGF-β1 expression significantly increased(t=76.01,P<0.001)in the keloid tissues compared with the expression levels of corresponding proteins in the normal tissues.CCK8 assay showed that the cell activity was significantly lower in the ROCK1 OE group than in the ROCK1 NC group after 24-hour transfection(t=3.25,3.78,P=0.031,0.019,respectively),and significantly higher in the shROCK1 group than in the sh NC group(t=3.12,2.79,P=0.036,0.049,respectively).Transwell assay s

关 键 词：瘢痕疙瘩 成纤维细胞 细胞增殖 细胞运动 转化生长因子β 钙黏着糖蛋白类 ROCK1基因 

分 类 号：R622[医药卫生—整形外科]