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组蛋白去乙酰化酶6通过调控成纤维细胞迁移和分化促进老年小鼠皮肤伤口愈合

秦玉梅¹, 李娉¹, 慕雪盼¹, 李志明¹, 孙宸¹, 薛文龙¹, 孙皎², 白姣姣², 朱依纯¹, 王铭洁^1,*

¹小分子活性物质上海市高校重点实验室，上海市老年医学临床重点实验室，上海市高水平地方高校创新团队，复旦大学基础医学院生理与病理生理学系，上海 200032；²复旦大学附属华东医院内分泌科，上海 200040

摘要

随着年龄的增长，皮肤伤口愈合缓慢，不利于日常生活中的伤口恢复和术后的恢复。本文旨在研究组蛋白去乙酰化酶6 (histone deacetylase 6, HDAC6)对老年皮肤伤口愈合的影响。以体外培养人真皮成纤维细胞(human dermal fibroblasts, HDFs)和小鼠全层皮肤创面模型为研究对象，用划痕损伤移实验评价HDFs的迁移能力，用BrdU掺入实验评估增殖速度，用Western blot检测I、III型胶原蛋白以及HDAC6表达情况，用HDAC6选择性抑制剂tubastatin A (TsA)抑制HDFs中HDAC6活性、siRNA降低蛋白表达后观察HDFs的功能变化。利用转化生长因子β1 (transforming growth factor β1, TGF-β1)诱导HDFs分化，观察HDAC6对HDFs分化的影响。制备小鼠背部皮肤伤口愈合模型，通过拍照统计第14天时小鼠皮肤伤口闭合率评价伤口愈合情况，用HE染色观察伤口恢复情况，用免疫组织化学法检测CD31、α-SMA、I、III型胶原蛋白表达情况，用Masson三色染色法检测皮肤伤口组织处胶原沉积，观察小鼠皮肤伤口愈合情况。结果显示，衰老HDFs迁移速度、增殖速度明显下降。衰老的HDFs、老年小鼠皮肤中胶原蛋白和HDAC6蛋白表达显著降低。用TsA抑制HDAC6活性和siRNA敲减HDAC6均可降低HDFs迁移速度，显著抑制TGF-β1诱导的HDFs分化。与野生型年轻小鼠相比，HDAC6基因敲除年轻小鼠伤口愈合明显延迟。老年小鼠皮肤伤口愈合速度显著慢于年轻小鼠，HDAC6基因敲除老年小鼠皮肤伤口愈合速度进一步下降。和野生型老年小鼠相比，HDAC6基因敲除老年小鼠皮肤伤口血管新生延迟、胶原合成减少，胶原沉积下降。上述结果提示，老年皮肤伤口愈合延迟与成纤维细胞功能受损有关，HDAC6的充分表达是成纤维细胞迁移和分化所必需。

关键词： 皮肤伤口愈合; 成纤维细胞迁移; 成纤维细胞分化; 衰老; 组蛋白去乙酰化酶6

Histone deacetylase 6 promotes skin wound healing by regulating fibroblast migration and differentiation in aged mice

QIN Yu-Mei¹, LI Ping¹, MU Xue-Pan¹, LI Zhi-Min¹, SUN Chen¹, XUE Wen-Long¹, SUN Jiao², BAI Jiao-Jiao², ZHU Yi-Chun¹, WANG Ming-Jie^1,*

¹Shanghai Key Laboratory of Bioactive Small Molecules and Shanghai Key Laboratory of Clinical Geriatric Medicine, the Innovative Research Team of High-level Local Universities in Shanghai, Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China；²Department of Endocrinology, Huadong Hospital Affiliated to Fudan University, Shanghai 200040, China

Abstract

Skin wound healing tends to slow down with aging, which is detrimental to both minor wound recovery in daily life and the recovery after surgery. The aim of current study was to explore the effect of histone deacetylase 6 (HDAC6) on wound healing during aging. Cultured human dermal fibroblasts (HDFs) and mouse full-thickness skin wound model were used to explore the functional changes of replicative senescent dermal fibroblasts and the effect of aging on skin wound healing. Scratch wound healing assay revealed significantly decreased migration speed of senescent HDFs, and BrdU incorporation assay indicated their considerably retardant proliferation. The protein expression levels of collagen and HDAC6 were significantly decreased in both senescent HDFs and skin tissues from aged mice. HDAC6 activity inhibition with highly selective inhibitor tubastatin A (TsA) or HDAC6 knockdown with siRNA decreased the migration speed of HDFs and considerably suppressed fibroblast differentiation induced by transforming growth factor-β1 (TGF-β1), which suggests the involvement of HDAC6 in regulating fundamental physiological activities of dermal fibroblasts. In vivo full-thickness skin wound healing was significantly delayed in young HDAC6 knockout mice when compared with young wild type mice. In addition, the wound healing was significantly slower in aged wild type mice than that in young wild type mice, and became even worse in aged HDAC6 knockout aged mice. Compared to the aged wild type mice, aged HDAC6 knockout mice exhibited delayed angiogenesis, reduced collagen synthesis, and decreased collagen deposition in skin wounds. Together, these results suggest that delayed skin wound healing in aged mice is associated with impaired fibroblast function. Adequate expression and activity of HDAC6 are required for fibroblasts migration and differentiation.

Key words: skin wound healing; fibroblast migration; fibroblast differentiation; aging; histone deacetylase 6

收稿日期：　　录用日期：

通讯作者：王铭洁　　E-mail: mjwang@shmu.edu.cn

引用本文：

秦玉梅, 李娉, 慕雪盼, 李志明, 孙宸, 薛文龙, 孙皎, 白姣姣, 朱依纯, 王铭洁. 组蛋白去乙酰化酶6通过调控成纤维细胞迁移和分化促进老年小鼠皮肤伤口愈合[J]. 生理学报 2022; 74 (6): 979-992.

QIN Yu-Mei, LI Ping, MU Xue-Pan, LI Zhi-Min, SUN Chen, XUE Wen-Long, SUN Jiao, BAI Jiao-Jiao, ZHU Yi-Chun, WANG Ming-Jie. Histone deacetylase 6 promotes skin wound healing by regulating fibroblast migration and differentiation in aged mice. Acta Physiol Sin 2022; 74 (6): 979-992