Zhao Xiaohong, Bian Ruyu, Wang Fan, Wang Ying, Li Xue, Guo Yicheng, Zhang Xiaorong, Luo Gaoxing, Zhan Rixing
Institute of Burn Research; State Key Laboratory of Trauma, Burn and Combined Injury; Southwest Hospital, The Third Military Medical University (Army Medical University), Chongqing, 400038, China.
Department of Plastic and Reconstructive Surgery, Southwest Hospital, The Third Military Medical University (Army Medical University), Chongqing, 400038, China.
Stem Cell Res Ther. 2021 Jan 7;12(1):42. doi: 10.1186/s13287-020-02106-7.
Epidermal stem cells (EpSCs) can self-renew, which are responsible for the long-term maintenance of the skin, and it also plays a critical role in wound re-epithelization, but the mechanism underlying EpSCs proliferation is unclear. GDF-5, also known as BMP-14, is a member of the BMP family and can be used as a self-renewal supporter. Here, we studied the effects of GDF-5 on mouse EpSCs proliferation mechanism in wound healing.
Firstly, the effects of GDF-5 on EpSCs proliferation was tested by using CCK8 reagent and PCNA expression was analyzed by Western blotting. Secondly, we screened genes that promote EpSCs proliferation in the FOX and cyclin family by qPCR, and then the protein expression level of the selected genes was further analyzed by Western blotting. Thirdly, siRNA plasmids and pAdEasy adenovirus were transfected or infected, respectively, into mouse EpSCs to detect the effect of target genes on GDF-5-induced cell proliferation. Furthermore, we injected GDF-5 to a deep partial thickness burn mouse model for finding out whether EpSCs proliferation can be detected by immunohistochemical. Finally, the relevant target genes were analyzed by qPCR, immunoblotting, and dual-luciferase reporter gene detection.
We discovered that 100 ng/ml recombinant mouse GDF-5 was the optimal concentration for promoting mouse EpSCs proliferation. Through preliminary screened by qPCR, we found that Foxg1 and cyclin D1 could be the downstream molecules of GDF-5, and the results were confirmed by Western blotting. And the effect of GDF-5 on mouse EpSCs proliferation was adjusted by Foxg1/cyclin D1 in vitro and in vivo. Besides, GDF-5-induced transcription of cyclin D1 was regulated by Foxg1-mediated cyclin D1 promoter activity.
This paper showed that GDF-5 promotes mouse EpSCs proliferation via Foxg1-cyclin D1 signal pathway. It is suggested that GDF-5 may be a new approach to make EpSCs proliferation which can be used in wound healing.
表皮干细胞（EpSCs）能够自我更新，负责皮肤的长期维持，并且在伤口再上皮化过程中也发挥关键作用，但其增殖的潜在机制尚不清楚。生长分化因子5（GDF-5），也称为骨形态发生蛋白14（BMP-14），是骨形态发生蛋白（BMP）家族的成员，可作为自我更新的支持因子。在此，我们研究了GDF-5对伤口愈合过程中小鼠EpSCs增殖机制的影响。
首先，使用CCK8试剂检测GDF-5对EpSCs增殖的影响，并通过蛋白质免疫印迹法分析增殖细胞核抗原（PCNA）的表达。其次，通过定量聚合酶链反应（qPCR）筛选FOX和细胞周期蛋白家族中促进EpSCs增殖的基因，然后通过蛋白质免疫印迹法进一步分析所选基因的蛋白质表达水平。第三，分别将小干扰RNA（siRNA）质粒和pAdEasy腺病毒转染或感染小鼠EpSCs，以检测靶基因对GDF-5诱导的细胞增殖的影响。此外，我们将GDF-5注射到深度部分厚度烧伤小鼠模型中，以确定是否可通过免疫组织化学检测到EpSCs增殖。最后，通过qPCR、免疫印迹和双荧光素酶报告基因检测对相关靶基因进行分析。
我们发现100 ng/ml重组小鼠GDF-5是促进小鼠EpSCs增殖的最佳浓度。通过qPCR初步筛选，我们发现叉头框蛋白G1（Foxg1）和细胞周期蛋白D1（cyclin D1）可能是GDF-5的下游分子，蛋白质免疫印迹法证实了该结果。并且在体外和体内，GDF-5对小鼠EpSCs增殖的影响由Foxg1/细胞周期蛋白D1调节。此外，GDF-5诱导的细胞周期蛋白D1转录受Foxg1介导的细胞周期蛋白D1启动子活性调控。
本文表明GDF-5通过Foxg1-细胞周期蛋白D1信号通路促进小鼠EpSCs增殖。提示GDF-5可能是一种促使EpSCs增殖的新方法，可用于伤口愈合。
