龟板散调控Wnt/BMP信号诱导毛囊干细胞防治压疮

Plastrum Testudinis Power (PTP), which has effective components including Plastrum Testudinis (PT) and Chinese Goldthread, is the representative recipe of clearing toxin and nourishing yin method, and might treat pressure ulcers. Pressure Ulcer is a major problem in clinical nursing. As a new viewpoint about skin repairing, hair follicle stem cell (HFSC) is a key target of treatment pressure ulcer and can be regulated by Wnt and bone morphogenetic protein (BMP) signal pathways to induce proliferation and differentiation. Our investigations showed that PT could regulate Wnt/BMP signal pathways of Mesenchymal stem cell (MSC) and promote epidermic stem cell (ESC) proliferation and antiapoptosis. This project postulates that PTP might induce HFSC proliferation and differentiation through Wnt/BMP signaling pathways to treat Pressure ulcer. The investigation constructs CDK4/Id1 promoters Dual-Luciferase report genes to research the mechanism of PT effect on HSFC cell generation cycle cell, and cultures HFSC in vitro with different culture mediums including PT, Wnt/BMP inhibitors or interfere by SiRNA and gene transfer to research the mechanism of PT regulating Wnt/BMP signaling pathway detected with ELISPOT, Flow Cytometer method etc. Pharmacological mechanism of PTP in vivo should be evaluated with BrdU and pathohistological technology through making pressure sores models of murine or swine. Plastrum Testudinis not only promotes HFSC proliferation via Wnt signal pathway but also leads to proliferated HFSC differentiation via BMP signal pathway. PTP has the security characteristic of nature medicine, maybe safer than some growth factors, such as bFGF. This project illuminates the molecular mechanism of PTP regulating Wnt/BMP signal pathway to induce HFSC, also prefers a further integrated theory of pressure ulcer based on Chinese Traditional Medicine and offers a new method for clinical nursing treatment pressure ulcer.

压疮是常见的皮肤损伤疾病，至今仍属于临床护理难题。Wnt/BMP以不同信号通路调控毛囊干细胞（HFSC）增殖与分化修复皮肤损伤是治疗压疮的关键，传统方药龟板散可防治压疮。前期研究表明龟板滋阴潜阳，可以调控Wnt/BMP信号，促表皮干细胞增殖，推测龟板散可能通过调控Wnt/BMP信号诱导HFSC增殖修复皮肤。本项目构建CDK4/Id1启动子驱动的双荧光素酶报告基因，研究龟板调控HFSC增殖机制；在体外培养HFSC结合siRNA和基因转染技术，以ELISPOT、流式细胞仪等研究龟板散调控Wnt/BMP分子机制；采用BrdU标记HFSC等技术在动物压疮模型上研究龟板散防治压疮药理作用。龟板不仅通过Wnt信号启动HFSC增殖，也通过BMP信号诱导增殖后的细胞分化，具有天然药物的安全性。本项目深入研究龟板散调控HFSC分子机制，为解决临床康复与护理难题提供理论基础，在皮肤疾病治疗方面有广泛应用前景。

本项目研究认为龟板有效成分促毛囊干细胞（HFSC）增殖效应是通过激活Wnt/β-catenin 通路、抑制BMP信号通路实现的。本项目按照研究计划，体外培养大鼠触须皮肤HFSC，采用CD34、CD71、CK15、Lgr5等表面标记物鉴定，分析了在龟板有效成分S8、S9对Wnt/β-catenin 和BMP信号通路各因子的蛋白和mRNA表达情况。S8、S9可上调β-catenin的表达，GSK-3β下降，β-catenin信号相关的转录因子和细胞周期蛋白Lef1、c-myc、cyclinD1表达升高，S8作用下Tcf3表达上升，S9作用下Tcf3表达下降；同时S8、S9下调BMP4的表达。.本项目研究改良了大鼠压疮模型，采用缺血12 h 再灌注12 h无创造模方法，成功构建了I期压疮皮损；在此模型基础上发现S8、S9及龟板总提取物对皮肤的压疮有较好的修复作用，与激活了创伤部位毛囊干细胞Wnt信号通路有关。为扩大龟板在皮肤损伤中的适应范围，项目组还建立了大鼠触须部皮肤创伤模型，结果表明S8组与S9组可以促进创面的愈合，证实S8、S9可以通过Wnt/β-catenin信号通路促进大鼠触须部创面愈合。本项目还进行了龟板有效成分诱导后HFSC的移植研究，将诱导后的HFSCs混悬液注入大鼠触须部创面，HE和VG染色结果表明，S9组大鼠创面皮肤出血少炎症反应轻，皮肤胶原纤维含量最为丰富。.本项目还根据细胞实验和动物实验结果，研制了一种用于治疗压疮的喷膜剂，并提交了专利申请书申请专利1项。该喷膜剂可以有效隔离细菌，促进肉芽组织生长，促进伤口修复，治疗早期压疮的有效率达95%以上，为今后的成果转化和临床应用打下了良好基础。.本项目已达到研究目标。现已经发表中文核心期刊文章1篇，已被录用SCI论文1篇和中文核心期刊论文1篇，培养硕士研究生2名，提交了发明专利申请1项，相关研究工作还在进行中。