中华皮肤科杂志 ›› 2023, Vol. 56 ›› Issue (3): 222-228.doi: 10.35541/cjd.20210675

• 论著 • 上一篇    下一篇

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

桑鹏飞1    方明松1    李旋1    曹林1    赵玲玲1    刘畅1    蒋智永1    朱飞2   

  1. 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
  • 收稿日期:2021-09-15 修回日期:2022-12-02 发布日期:2023-03-06
  • 通讯作者: 朱飞 E-mail:hfzfzx@163.com
  • 基金资助:
    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）

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

Sang Pengfei1, Fang Mingsong1, Li Xuan1, Cao Lin1, Zhao Lingling1, Liu Chang1, Jiang Zhiyong1, Zhu Fei2   

  1. 1Department of Plastic Surgery, The Second People′s Hospital of Hefei （Hefei Hospital Affiliated to Anhui Medical University）, Hefei 230041, China; 2Department of Plastic Surgery, The First Affiliated Hospital of Anhui Medical University, Hefei 230022, China
  • Received:2021-09-15 Revised:2022-12-02 Published:2023-03-06
  • Contact: Zhu Fei E-mail:hfzfzx@163.com
  • Supported by:
    Provincial Natural Science Research Key Project of Anhui Universities （KJ2014A108）; Anhui Medical University Research Fund Project （2019xkj094）

PDF

1

摘要： 【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。

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

Abstract: 【Abstract】 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 factor β1 （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 showed that the number of migratory cells was significantly lower in the ROCK1 OE group than in the ROCK1 NC group （t = 5.17, P = 0.004）, and significantly higher in the shROCK1 group than in the sh NC group （t = 9.28, P < 0.001）. Compared with the ROCK1 NC group, the ROCK1 OE group showed significantly increased mRNA and protein expression levels of ROCK1 and E-cadherin （P < 0.05 or < 0.001）, but decreased mRNA and protein expression levels of TGF-β1 （both P < 0.001）; compared with the sh NC group, the shROCK1 group showed significantly decreased mRNA and protein expression levels of ROCK1 and E-cadherin （P < 0.05 or < 0.001）, but significantly increased mRNA and protein expression levels of TGF-β1 （P = 0.005 or < 0.001）. Conclusions The ROCK1 gene can inhibit the proliferation and migration of HKFs. Overexpression of the ROCK1 gene can down-regulate the TGF-β1 gene expression and up-regulate the E-cadherin gene expression in HKFs.

Key words: Keloid, Fibroblasts, Cell proliferation, Cell movement, Transforming growth factor beta, Cadherins, Gene, ROCK1

引用本文

桑鹏飞 方明松 李旋 曹林 赵玲玲 刘畅 蒋智永 朱飞. 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[J]. 中华皮肤科杂志, 2023,56(3):222-228. doi:10.35541/cjd.20210675

Sang Pengfei, Fang Mingsong, Li Xuan, Cao Lin, Zhao Lingling, Liu Chang, Jiang Zhiyong, Zhu Fei. Effects of the ROCK1 gene on proliferation and migration of and related molecular expression in keloid fibroblasts[J]. Chinese Journal of Dermatology, 2023, 56(3): 222-228.doi:10.35541/cjd.20210675