c-Cbl调控MSCs脂筏内外CXCR4异常分布在糖尿病创面血管化障碍中的作用及机制研究

The chronic wound of diabetes mellitus (DM) has difficult in healing and treatment. The decreased functions of endogenous MSCs (mesenchymal stem cell, MSCs) have been regarded as an important factor in wound vascularization disorder and delayed healing. The mechanism of dysregulation of SDF-1/CXCR4, a key signaling axis of MSCs, which could lead to wound vascularization disorder under the DM state, needs further study. Studies have shown that CXCR4 needs to be localized to the lipid raft microdomains in order to activate downstream signals. In addition, c-Cbl which acts as an E3 ubiquitin ligase was found to promote lipid raft depolymerization and down-regulate cell signal transduction mediated by related membrane receptors. Pre-test found that DM mouse serum-stimulated abnormal expression of CXCR4 both inside and outside the MSCs lipid raft, meanwhile the phosphorylation level of c-Cbl was abnormally increased in MSCs. All above indicating that abnormal regulation of CXCR4 by c-Cbl may be the key role in decreased pro-angiogenic function of MSCs. In this study, models of DM wound and MSCs will be established. Total Internal Reflection Fluorescence Microscopy, Single Particle Tracking and other conventional assays will be applied to dynamic observe and identify the role and mechanism of c-Cbl regulating the abnormal expression of CXCR4 both inside and outside the MSCs lipid raft in DM wound vascularization disorder, thus providing a new target for the treatment of refractory wounds in DM.

糖尿病（DM）慢性创面迁延不愈、防治困难，内源性MSCs功能降低是创面血管化障碍并导致创面难愈的重要因素。DM状态下MSCs的关键信号轴SDF-1/CXCR4调控功能受损并导致创面血管化障碍的机制亟待深入研究。研究表明CXCR4需要定位在脂筏微结构域上才能激活下游信号。另外发现c-Cbl作为一种E3泛素连接酶，可促进脂筏解聚并下调相关膜受体介导的细胞信号转导。前期预试验发现DM小鼠血清刺激下MSCs脂筏内外CXCR4的表达异常，同时MSCs中c-Cbl磷酸化水平也异常增高，表明c-Cbl调控CXCR4异常分布可能是MSCs促血管生成功能减弱的关键因素。本课题拟建立DM创面小鼠和MSCs模型，采用全内反射单分子荧光显微成像技术、单粒子追踪技术及其他常规试验方法，动态观察并阐明c-Cbl调控MSCs脂筏内外CXCR4异常分布在DM创面血管化障碍中的作用及机制，从而为DM难愈创面治疗提供新靶点。

糖尿病（diabetes mellitus，DM）的发病率逐年上升，而糖尿病创面迁延不愈是糖尿病严重的并发症之一，其中以糖尿病足最为常见，其中14~20%的糖尿病足最终将发展为糖尿病足坏疽，导致患者截肢致残。虽然目前已有大量关于糖尿病难愈性创面的研究，但是已有的研究结果仍不能为其临床防治提供有效措施。本课题通过建立DM创面大鼠和MSCs模型并进行相关动物及细胞实验，动态观察了BM-MSCs在糖尿病外环境下CXCR4在细胞膜脂筏中的分布规律及变化特点；并深入研究c-Cbl表达变化对CXCR4在脂筏内外分布以及对下游Akt相关信号的作用及机制。发现通过抑制c-Cbl表达可以改善体外长期扩增培养BM-MSCs的增殖、迁移及旁分泌功能，进一步通过动物实验验证了“抑制c-Cbl表达可提高体外长时间扩增培养的BM-MSCs促进糖尿病创面血管生成及愈合的效果”，为糖尿病慢性创面的防治策略提供了新的思路，同时也为干细胞治疗的优化提供了重要的依据。另外，在通过动物实验研究糖尿病创面的过程中，我们还发现相对于生理盐水，局部使用富氢生理盐水明显缩短了创面闭合时间、提高了创面愈合率，并且发现其作用的机制可能是通过Nrf-2/HO-1信号通路抑制氧化应激反应实现的。本项目所获相关研究结果进一步深化了对糖尿病创面血管生成障碍机制的认识，为糖尿病创面的临床防治提供了新的思路及潜在的治疗靶点，具有一定的临床转化价值。