回阳生肌内外合治法对慢性皮肤溃疡（阴证）愈合过程中MSCs趋化迁移及分泌功能的调节作用研究

Mesenchymal stem cells (MSCs) have been defined as the ‘core ’ of the wound healing process, and delayed healing wound is closely related with impaired MSCs proliferation, migration and secretion. Chronic wound with Yin syndrom is a refractory disease. In Traditional Chinese Medicine, tonifying kidney to replenish essence and invigorating spleen and replenishing qi are the principle for ‘rescuing Yang and improving tissue regeneration’, which is effective in clinical for improving wound healing. Previous research in our lab has confirmed that HuiYangShengJi decoction could improve proliferation and migration of MSCs. Based on the clinical experience and previous research, we try to explore oral and topical administration ‘rescuing Yang and improving tissue regeneration’ therapy on MSCs’ function such as its homing and secretion during chronic wound healing process(Yin syndrome) by tissue biopsy and animal models. A comparative study is designed to observe single oral use, single topical use and both of them on bone marrow MSCs and wound MSCs function and migration in diabetic mice and immunodepressive mice wound model. In vitro, bone marrow MSCs gene expression microarrays from diabetic mice is analysed to study both oral and topical herbal medicine extracts on differential gene-mediated signaling pathways, in order to clarify the targets of oral and/or topical use medicine of ‘rescuing Yang and improving tissue regeneration’. Via clinical and animal experiment in vivo, and MSCs culture in vitro, we try to explore the mechanism of ‘rescuing Yang and improving tissue regeneration’ therapy on treating delayed wound healing(Yin syndrome) as to provide the biological evidence for clinical application.

间充质干细胞（MSCs）已被定义为创面愈合过程的“核心”，慢性皮肤溃疡愈合迟缓与MSCs增殖、迁移及分泌功能受损密切相关。慢性皮肤溃疡阴证是临床难治证候，以补肾填精、健脾益气为治则的回阳生肌法通过内外合治治疗慢性皮肤溃疡阴证有良好疗效。前期研究发现回阳生肌汤可促进MSCs增殖和迁移。以此为线索，本项目通过临床样本检测及动物实验，明确回阳生肌内外合治法对创面愈合及MSCs功能的调节作用。以回阳生肌内外合治法、单用内治法、单用外治法进行对比研究，观察药物对糖尿病小鼠及免疫抑制小鼠复合慢性伤口模型的骨髓MSCs功能、迁移至伤口局部的MSCs功能的调节作用，探索其作用环节。通过糖尿病小鼠来源的骨髓MSCs体外模型结合表达谱芯片检测，观察回阳生肌内外合治法对“差异基因→MSCs功能调节通路”的干预作用，阐明其作用靶点。通过临床、动物、细胞多层次的研究，为回阳生肌内外合治法的临床应用提供生物学证据。

慢性皮肤溃疡阴证属于阴疮，是临床难治证候。阴疮不仅表现在局部的修复障碍，也与骨髓间充质干细胞（BMSCs）增殖、迁移及分泌功能受损密切相关。BMSCs在创面愈合过程发挥“核心”作用，具有中医“肾精”的属性，通过迁移分化在创面发挥修复作用，体现了精化气，精血互生，发挥回阳生肌的功能。以补肾填精、健脾益气为治则的回阳生肌法通过内外合治治疗慢性皮肤溃疡阴证有良好疗效。本研究通过回阳生肌内外合治法对慢性皮肤溃疡（阴证）患者创面、免疫抑制小鼠、糖尿病db/db小鼠和骨髓抑制大鼠愈合迟缓皮肤伤口和体外间充质干细胞模型进行研究，证实：1.回阳生肌内治法、外治法、合治法均能促进慢性创面愈合；2.内治法的差异蛋白主要为生长因子，包括IGF-Ⅰ、EGFR、HGF，其参与的信号通路也多与生长因子有关；而外治法的差异蛋白主要为趋化因子，包括M-CSF、CCL22、CCL25 CCL27、CXCL11等，其参与的信号通路也多为趋化因子相关通路；合治组的差异蛋白为M-CSF、TGF-β1、VEGFR1，包括趋化因子和生长因子，参与了骨髓造血和创面修复细胞的增殖和迁移。3.在db/db小鼠复合伤口模型上证实回阳生肌汤内治可促进db/db小鼠骨髓干细胞的增殖，增加创面MSCs细胞的数量，调控创面的炎症反应，促进肉芽组织新生，从而加速创面愈合。4.在骨髓抑制大鼠愈合迟缓皮肤伤口模型上证实回阳生肌汤可升高大鼠血清中SDF-1和G-CSF的浓度，提升外周血白细胞数量和骨髓、外周血中BMSCs、HSCs数量和创面HSCs数量，且大鼠骨髓BMSCs的增殖、趋化迁移能力增强，提示其通过激活SDF-1/CXCR4轴促进骨髓干细胞的增殖、趋化迁移至创面来促进创面修复。5.体外间充质干细胞氧化应激模型证实回阳生肌汤和鸡血藤通过激活SDF-1/CXCR4信号轴，保护氧化损伤BMSCs的增殖活性，提升氧化应激状态下BMSCs的迁移和趋化能力。6.回阳生肌汤和回阳生肌膏联合治疗慢性皮肤溃疡患者（阴证）4周后，患者下肢肿胀明显减轻，创周皮肤颜色变淡，创面颜色红活，创面面积缩小。课题从临床、动物、体外实验三个层面阐明回阳生肌内外合治法治疗慢性皮肤溃疡（阴证）的作用机制，体现了内治法的重要性，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。