自制毛囊再生乳膏对小鼠皮肤创面毛囊再生的影响

Effect of self-made hair follicle regeneration ointment on hair follicle regeneration in mouse skin wound

背景创面愈合时无毛囊等皮肤附属器再生是创面修复领域的难题。小分子化合物能够调控细胞增殖与分化,促使成熟体细胞再分化为具有增殖能力的前体细胞或其他细胞系,还能够靶向调控信号通路与代谢进程,是一种促进皮肤及附属器原位再生的安全有效的方法。目的本研究拟通过小分子化合物组合探索其促进创面毛囊再生的效果及机制。方法选取C57BL/6小鼠45只,雌性,体质量(20±5)g。以打孔器配合眼科剪制作小鼠背部双侧皮肤全层缺损创面动物模型,左侧创面为用药侧,右侧创面为对照侧。以负载CHIR-99021、Purmorphamine、Tofacitinib和SJ000291942四种小分子化合物的凡士林药膏(以下简称毛囊再生乳膏)连续涂抹小鼠背部皮肤全层缺损创面28 d,通过常规HE染色、Masson染色以及免疫组织化学染色分析创面愈合和毛囊再生情况。结果用药组与对照组小鼠背部创面愈合率无统计学差异,提示毛囊再生乳膏对创面愈合速度无显著影响。HE染色发现,毛囊再生乳膏处理创面14 d左右,用药组创面表皮形成毛囊上皮芽基细胞,真皮中出现毛乳头细胞。28 d可见用药组创面中大量毛囊样结构,免疫荧光染色证实其表达毛囊细胞特征性标志物CK5、CK15、CK17、Noggin,而对照组创面毛囊再生数量极少,用药组与对照组新生毛囊数量差异有统计学意义(17 vs 4,P<0.001)。Masson染色可见用药组与对照组创面胶原纤维密度及排列方式无明显区别,提示毛囊再生乳膏可诱导纤维瘢痕中毛囊再生。免疫组织化学染色显示用药侧创面新生的表皮细胞、毛囊上皮芽基细胞、新生毛乳头细胞中Wnt信号通路和Shh信号通路相关标志物β-catenin、Shh、Gli1显色明显,表明毛囊再生乳膏可能通过激活Wnt、Shh信号通路促进创面毛囊再生。结论本研究自制的毛囊再生乳膏能够通过调控毛囊生长发育过程中的Wnt信号通路和Shh信号通路促进创面愈合时毛囊再生,有望转化为创面治疗药物应用于临床。

Background The absence of hair follicles and other skin appendages regeneration is a difficult problem in the field of wound healing,especially cicatricial alopecia caused by scalp(burn)injury,which seriously affects the life and work of patients.Small molecule compounds can regulate cell proliferation and differentiation,promote the redifferentiation of mature cells into proliferative precursor cells or other cell lines,and also target the regulation of signaling pathways and metabolic processes,which is a safe and effective method to promote the in situ regeneration of skin and skin appendages.Objective To explore the effect and mechanism of promoting hair follicle regeneration in mouse wound through the combination of small molecule compounds.Methods Forty-five female C57 BL/6 mice with a weight of(20±5)g were selected in this study.The animal model of fullthickness skin defect on both sides of the back of mice was made with perforator and ophthalmic scissors.The left side of the wound was the drug side,and the right side of the wound was the control side.We prepared the Vaseline ointment containing the small molecules CHIR-99021,Purmorphamine,Tofacitinib,SJ000291942 and applied the ointment on the mouse skin wounds for 28 days.HE staining,Masson staining,and immunohistochemistry were performed to analyze the condition of wound healing and hair follicle regeneration.Results There was no significant difference in the healing rate between the treatment group and the control group,suggesting that hair follicle regeneration ointment had no significant effect on the healing rate of the wound.HE staining showed that about 14 days after treatment of the wound with hair follicle regeneration ointment,hair germ cells were formed on the wound epidermis and dermal papilla cells were found in the treatment group.On the 28 th day,a large number of follicle-like structures could be seen on the wound surface,which was confirmed by immunofluorescence staining to express characteristic markers of hair follicle cells(CK5,CK15,CK17 and Noggin).However,the number of hair follicles regenerated after applying ointment alone was very small,and there was a significant statistical difference in the number of new hair follicles between the treatment group and the control group(17 vs 4,P<0.001).Masson staining showed that there was no significant difference in the density and arrangement of collagen fibers between the treatment group and the control group,suggesting that hair follicle regeneration ointment could induce hair follicle regeneration in fiber scar.Immunohistochemical staining showed that markers related to Wnt signaling pathway and sonic hedgehog(Shh)signaling pathway were obvious in the newly formed epidermal cells,hair germ cells,and newly formed dermal papilla cells on the drug side,suggesting that hair follicle regeneration ointment might promote hair follicle regeneration on the wound by activating Wnt and Shh signaling pathway.Conclusion In this study,the selfmade hair follicle regeneration ointment can promote hair follicle regeneration during wound healing by regulating the Wnt signaling pathway and Shh signaling pathway,which is expected to be transformed into a wound therapy drug for clinical application.