中华皮肤科杂志 ›› 2021, Vol. 54 ›› Issue (3): 196-200.doi: 10.35541/cjd.20200807

• 论著 • 上一篇    下一篇

张力促进瘢痕疙瘩纤维化的初步研究

宋海峰    董高宏    魏凯军    胡新红    张衍国    刘涛   

  1. 空军军医大学唐都医院皮肤科,西安  710038
  • 收稿日期:2020-08-14 修回日期:2020-12-28 发布日期:2021-03-02
  • 通讯作者: 刘涛;张衍国 E-mail:Morlab2@fmmu.edu.cn; zhangya3341@163.com
  • 基金资助:
    陕西省社会发展科技攻关项目(2015SF164)

Tension promotes keloid fibrosis: a preliminary study

Song Haifeng, Dong Gaohong, Wei Kaijun, Hu Xinhong, Zhang Yanguo, Liu Tao   

  1. Department of Dermatology, Tangdu Hospital, The Fourth Military Medical University, Xi′an 710038, China
  • Received:2020-08-14 Revised:2020-12-28 Published:2021-03-02
  • Contact: Liu Tao; Zhang Yanguo E-mail:Morlab2@fmmu.edu.cn; zhangya3341@163.com
  • Supported by:
    Science and Technology Development Projects of Shaanxi province(2015SF164)

摘要: 【摘要】 目的 初步探讨张力刺激对瘢痕疙瘩成纤维细胞生物学活性及纤维化标志基因表达的影响。方法 选取2017年1 - 3月于空军军医大学唐都医院皮肤科诊断为瘢痕疙瘩并接受手术治疗的3例患者,处理手术切除的瘢痕疙瘩组织,经原代培养获得人瘢痕疙瘩成纤维细胞(KD-Fbs),取3~6代KD-Fbs分别培养于张力刺激模型小室和对照小室中,分别接受张力刺激(张力组)和正常培养(对照组)。CCK8法检测培养1、2、3、4 d时KD-Fbs的增殖活性,划痕实验检测培养1、2 d时KD-Fbs的迁移能力;处理48 h后,实时定量PCR和Western印迹法分别检测KD-Fbs中纤维化标志物Ⅰ型胶原蛋白、纤连蛋白、α平滑肌肌动蛋白(α-SMA)mRNA和蛋白的表达。两组间比较采用两独立样本t检验。结果 CCK8法显示,培养1、2、3、4 d时,张力组KD-Fbs增殖活性均高于对照组(t值分别为3.05、7.00、16.65、15.19,均P < 0.05)。划痕实验显示,培养1、2 d时,张力组KD-Fbs迁移率(48.65% ± 3.96%、100.00%)均显著高于对照组(9.36% ± 1.14%、50.35% ± 4.23%;t值分别为16.53、20.35,均P < 0.01)。实时定量PCR结果显示,张力组Ⅰ型胶原蛋白、纤连蛋白、α-SMA mRNA表达(3.04 ± 0.20、2.16 ± 0.10、3.76 ± 0.24)均显著高于对照组(1.00),差异有统计学意义(t值分别为17.57、21.01、20.25,均P < 0.01)。Western印迹法显示,3种纤维化标志物蛋白表达水平变化与其mRNA变化一致(均P < 0.05)。结论 张力可能通过促进纤维化标志基因的表达,增强KD-Fbs的增殖和迁移能力,进而参与瘢痕疙瘩的纤维化进程。

关键词: 瘢痕疙瘩, 纤维化, 细胞增殖, 细胞迁移, 张力

Abstract: 【Abstract】 Objective To preliminarily evaluate the effect of tension stimulation on the biological activity of and expression of fibrosis marker genes in keloid fibroblasts (KD-Fbs). Methods Three patients who were diagnosed with keloids and received surgical treatment were collected from the Department of Dermatology, Tangdu Hospital, the Fourth Military Medical University from January to March 2017. Human KD-Fbs were isolated from resected keloid tissues, and subjected to primary culture. The third- to sixth-passage KD-Fbs were divided into tension group and control group to be cultured in the tension-based chamber and control chamber respectively, and subjected to tension stimulation and normal culture respectively. Cell counting kit-8 (CCK8) assay was performed to assess the proliferative activity of KD-Fbs after 1-, 2-, 3- and 4-day culture, and the scratch assay to evaluate the migratory ability of KD-Fbs after 1- and 2-day culture. After 48-hour treatment, real-time quantitative PCR and Western blot analysis were performed to determine the mRNA and protein expression of fibrosis markers typeⅠcollagen, fibronectin and α-smooth muscle actin (α-SMA) in KD-Fbs respectively. Two-independent-sample t test was used for comparisons between 2 groups. Results CCK8 assay showed that the proliferative activity of KD-Fbs was significantly higher in the tension group than in the control group after 1-, 2-, 3- and 4-day culture (t = 3.05, 7.00, 16.65, 15.19, respectively, all P < 0.05). After 1- and 2-day culture, the scratch assay showed that the migration rate of KD-Fbs was significantly higher in the tension group (48.65% ± 3.96%, 100.00%, respectively) than in the control group (9.36% ± 1.14%, 50.35% ± 4.23%, t = 16.53, 20.35, respectively, both P < 0.01). Real-time quantitative PCR showed that the mRNA expression of typeⅠcollagen, fibronectin and α-SMA was significantly higher in the tension group (3.04 ± 0.20, 2.16 ± 0.10, 3.76 ± 0.24, respectively) than in the control group (1.00; t = 17.57, 21.01, 20.25, respectively, all P < 0.01). As Western blot analysis revealed, changes in the protein expression of the 3 fibrosis markers were consistent with their mRNA expression changes (all P < 0.05). Conclusion Tension may participate in the fibrosis in keloids by promoting the expression of fibrosis marker genes, and enhancing the proliferative and migratory ability of KD-Fbs.

Key words: Keloid, Fibrosis, Cell proliferation, Cell migration assays, Tension