中华皮肤科杂志 ›› 2020, Vol. 53 ›› Issue (3): 184-189.doi: 10.35541/cjd.20190568

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SIRT6对老年人皮肤成纤维细胞迁移能力和增殖能力的影响及机制研究

翟晓艳1    裴亮2    赵焕新3    杨李旺1    杨蓉3    季新燕4   

  1. 1山西中医药大学解剖教研室，山西榆次  030619；2山西医科大学第二医院泌尿外科，
    太原  030001；3山西中医药大学生理教研室 山西榆次  030619；4山西中医药大学药理教研室 山西榆次  030619
  • 收稿日期:2019-05-14 修回日期:2019-12-20 发布日期:2020-03-03
  • 通讯作者: 翟晓艳 E-mail:zxy2005019@126.com
  • 基金资助:
    山西省应用基础研究计划项目（201901D211533）；山西中医药大学课题（2018PY-002）

Effect of SIRT6 on migratory and proliferative activity of skin fibroblasts in the elderly and their mechanisms

Zhai Xiaoyan1, Pei Liang2, Zhao Huanxin3, Yang Liwang1, Yang Rong3, Ji Xinyan4   

  1. 1Department of Anatomy, Shanxi University of Chinese Medicine, Yuci 030619, Shanxi, China; 2Department of Urologic Surgery, Second Hospital of Shanxi Medical University, Taiyuan 030001, China; 3Department of Physiology, Shanxi University of Chinese Medicine, Yuci 030619, Shanxi, China; 4Department of Pharmacology, Shanxi University of Chinese Medicine, Yuci 030619, Shanxi, China 
  • Received:2019-05-14 Revised:2019-12-20 Published:2020-03-03
  • Contact: Zhai Xiaoyan E-mail:zxy2005019@126.com
  • Supported by:
    Fibroblasts; Cell proliferation; Cell movement; NF-kappa B; Aged; Wound healing; SIRT6
    Fund programs: Applied Basic Research Program of Shanxi Province （201901D211533）; Program of Shanxi University of Chinese Medicine（2018PY-002）

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摘要： 目的 探讨沉默信息调节因子2同源类似物6（SIRT6）对老年人皮肤成纤维细胞迁移能力和增殖能力的影响及机制。方法 在山西医科大学第二附属医院泌尿外科取不同年龄患者包皮环切术切取的包皮组织，其中老年组8例，青年组8例。用胶原酶消化法提取人皮肤成纤维细胞，Western印迹法检测不同年龄组人皮肤成纤维细胞中SIRT6和磷酸化p65蛋白（p-p65）的表达，划痕实验检测细胞迁移活性，CCK8法检测细胞增殖活性。将老年组皮肤成纤维细胞分成两组，一组用SIRT6慢病毒感染使其SIRT6表达增高作为SIRT6组，另一组以空病毒感染作为对照组，用上述方法分别检测SIRT6组和对照组细胞迁移活性、增殖活性和p-p65蛋白表达水平，实时PCR检测两组Ⅰ型和Ⅲ胶原蛋白、整合素亚基α3、α5和β1 mRNA的表达。采用GraphPad Prism 5软件进行统计学分析，t检验分析两组之间数据的差异。结果 老年组皮肤成纤维细胞SIRT6表达水平（0.434 ± 0.179）显著低于青年组（1.000 ± 0.067，t = 3.040，P = 0.012），p-p65表达水平（1.694 ± 0.148）显著高于青年组（1.000 ± 0.093， t = 2.949，P = 0.015），迁移率（43.81% ± 18.84%）显著低于青年组（94.63% ± 12.32%，t = 5.903，P = 0.003），24、48 h增殖活性也较青年组显著下降（P < 0.05）。老年组成纤维细胞过表达SIRT6后，与对照组相比，p-p65表达显著下降（P < 0.05），迁移能力和增殖能力显著提高（P < 0.05），同时Ⅲ胶原蛋白和整合素亚基α3、α5和β1的mRNA表达水平显著升高（P < 0.05）。结论 SIRT6可提高老年人成纤维细胞的迁移能力和增殖能力，其机制可能与抑制NF-κB通路有关。

关键词: 成纤维细胞； 细胞增殖； 细胞运动； NF-κB； 老年人； 伤口愈合; , SIRT6

Abstract: Objective To assess the effect of silent mating type information regulation 2 homolog 6（SIRT6） on migratory and proliferative activity of skin fibroblasts in the elderly, and to explore their mechanisms. Methods Circumcised foreskins were obtained from patients of different ages in Department of Urologic Surgery, Second Hospital of Shanxi Medical University, including 8 elderly patients and 8 young patients. Human skin fibroblasts were isolated from the foreskin tissues by using a collagenase digestion method. Western blot analysis was performed to determine the expression of SIRT6 and phosphorylated p65 （p - p65） in human skin fibroblasts in different age groups, scratch assay to evaluate cell migratory activity, and cell counting kit-8 （CCK8） assay to assess cellular proliferative activity. Skin fibroblasts in the elderly group were divided into 2 groups: SIRT6 group transfected with a lentiviral vector overexpressing SIRT6, and control group transfected with an empty lentiviral vector. Then, the cell migratory and proliferative activity as well as p-p65 expression were measured by the above methods, and the mRNA expression of type Ⅰ and Ⅲ collagens, and integrin subunits α3, α5 and β1 was determined by real-time PCR in the SIRT6 group and control group. Statistical analysis was carried out with GraphPad Prism 5 software by using t test for comparison between 2 groups. Results Compared with the young group, the elderly group showed significantly decreased SIRT6 expression in skin fibroblasts （0.434 ± 0.179 vs. 1.000 ± 0.067, t = 3.040, P = 0.012）, migration rate （43.81% ± 18.84% vs. 94.63% ± 12.32%, t = 5.903, P = 0.003）and cellular proliferative activity at 24 and 48 hours（both P < 0.05）, but significantly increased p-p65 expression（1.694 ± 0.148 vs. 1.000 ± 0.093, t = 2.949, P = 0.015）. Compared with the control group, the SIRT6 group showed significantly decreased p-p65 expression（P < 0.05）, but significantly increased migratory and proliferative activity （both P < 0.05）, and elevated mRNA expression of type Ⅲ collagen and integrin subunits α3, α5 and β1（all P < 0.05）. Conclusion SIRT6 can improve the migratory and proliferative activity of human fibroblasts in the elderly, possibly by inhibiting the nuclear factor-κB pathway.

Key words: Fibroblasts, Cell proliferation, Cell movement, NF-kappa B, Aged, Wound healing, SIRT6

引用本文

翟晓艳 裴亮 赵焕新 杨李旺 杨蓉 季新燕. SIRT6对老年人皮肤成纤维细胞迁移能力和增殖能力的影响及机制研究[J]. 中华皮肤科杂志, 2020,53(3):184-189. doi:10.35541/cjd.20190568

Zhai Xiaoyan, Pei Liang, Zhao Huanxin, Yang Liwang, Yang Rong, Ji Xinyan. Effect of SIRT6 on migratory and proliferative activity of skin fibroblasts in the elderly and their mechanisms[J]. Chinese Journal of Dermatology, 2020, 53(3): 184-189.doi:10.35541/cjd.20190568