回阳生肌膏对慢性皮肤溃疡（阴疮）血管新生MicroRNA200b-GATA2-VEGFR2调控的影响

Chronic skin ulcer with Yin syndrome is the difficulty in the field of dermatologic surgery. “Rescuing Yang and improving tissue regeneration’ is the basic principle, which showed positive effect in improving chronic wound healing by typical prescription of ‘Hui Yang Sheng Ji’ ointment. The former results showed that ‘Hui Yang Sheng Ji’ ointment promoted impaired wound healing by modulating angiogenesis .MiRNA200b-GATA2-VEGFR2 signaling axis is disrupted in chronic wounds. This program propose to explore the regulation of ‘Hui Yang Sheng Ji’ ointment on miRNA200b-GATA2-VEGFR2 signaling axis in diabetic (db/db )wounds and in vitro human dermal microvessel endothelial cells with TNF-α induced\miRNA200b mimic transfected\GATA2 or VEGFR2 knockdown , observe endothelial cell proliferation, migration, lumen formation and microvessels sprouting with SiRNA, real- time PCR and Western blot method, reveal the " rescuing Yang ", " improving tissue regeneration " scientific connotation, provide theoretical basis for clinical treatment of chronic skin ulcers with Yin syndrome

慢性皮肤溃疡属于中医“疮疡”的阴疮，是当前皮肤外科领域亟待解决的问题。回阳生肌法是治疗阴疮的基本治则，回阳生肌膏是其代表方剂，临床疗效显著，研究其回阳、生肌的作用环节和机制倍受中医皮外科的关注。研究发现慢性创面miRNA200b-GATA2-VEGFR2信号轴异常，导致血管新生障碍是慢性皮肤溃疡的关键环节，我们以往研究证实回阳生肌膏能促进创面血管生成。本课题以糖尿病(db/db)小鼠皮肤伤口模型和TNF-α诱导、miRNA200b模拟物转染、GATA2 /VEGFR2敲低的血管内皮细胞为对象，观察回阳生肌膏对血管内皮细胞增生、迁移、微管腔形成和微血管出芽的影响，用SiRNA技术、荧光定量PCR和Western blot等方法，研究血管新生过程中miRNA200b-GATA2-VEGFR2信号轴调控变化规律，揭示“回阳”、“生肌”的科学内涵，为临床治疗阴证疮疡提供理论依据。

糖尿病性皮肤溃疡（阴证疮疡）miRNA200b-GATA2-VEGFR2信号轴的异常，导致创面血管新生异常，延缓了创面肉芽组织生成，从而延缓创面愈合。本课题通过回阳生肌法有效方剂回阳生肌膏，对糖尿病小鼠伤口愈合迟缓模型和体外皮肤血管内皮细胞为模型进行研究。结果显示回阳生肌膏可缩小模型小鼠慢性创面面积，减轻创面炎症反应；促进血管新生从而促进创面愈合；治疗后7天可下调创面局部microRNA200b水平；14天时可升高VEGFR2和GATA2 mRNA和蛋白水平；10天时升高创面局部血流量。体外实验发现回阳生肌膏可促进TNF-α诱导的血管内皮细胞增殖、迁移和管腔形成；可以下调TNF-α诱导的血管内皮细胞miRNA200b水平，升高GATA2 和VEGFR-2蛋白表达；可以促进miRNA200b模拟物转染的血管内皮细胞GATA2 和VEGFR-2蛋白表达。通过以上研究证实回阳生肌膏可通过调控miRNA200b-GATA2-VEGFR2信号轴来促进创面血管新生，促使创面由炎症期进入增生期，是其促进慢性创面愈合的主要作用机制。. 本研究从体内外研究，以血管新生异常为靶点，揭示 “回阳”和 “生肌”的靶点，提升回阳、生肌的科学内涵，为提升回阳生肌法的科学内涵和临床应用提供了理论指导。