Semaphorin4D/CD100调节糖尿病慢性创面愈合的作用及机制

Diabetic chronic wound is demonstrated to be an increasing public-health problem nowadays. Unfortunately, treatment provided for diabetic chronic wound is often inadequate. Many evidences indicate that the chronic ischemia and hypoxia condition resulting from the impaired microcirculation correlated well with the pathogenesis of chronic wounds, however, the molecular mechanisms involved have not been fully elucidated. CD100, a kind of immune semaphorin, was demonstrated to play an important role in normal wound healing process. Our previous studies and researches in other groups showed that CD100 could promote effective angiogenesis not only in vivo but also in vitro. In our previous preliminary clinical studies, CD100 expression in diabetic foot ulcers was significantly lower than that in acute wounds. But it was still unknown that whether the CD100 expression correlated with the pathogenesis of diabetic foot ulcer and whether the addition of exogenous soluble CD100 could accelerate chronic wound healing by promoting angiogenesis. Based on our previous studies, this research intends to expand the diabetic foot sample collection and analyze the relationship between the expression of CD100 and its receptor and the wound healing course and the disease prognosis. The diabetic mouse chronic wound healing model and the hypoxia and high glucose endothelial culture model would also be treated with exogenous soluble CD100 or the CD100 receptor blocking reagents for regulating CD100 and its receptor signals, and then the impact of different treatment on wound healing process, angiogenesis and endothelial function will be evaluated. Through this research, the role and mechanism of CD100 and its receptor involving in chronic wound healing process would be confirmed, the results might provide potential new therapeutic target to chronic wounds healing.

糖尿病慢性创面是外科经常遇到的难题，目前在临床上仍缺乏有效的治疗方法。血管病变引起的组织细胞慢性缺血缺氧是其发生的重要原因，但分子机制尚未完全阐明。文献报道和我们前期研究表明，免疫信号蛋白CD100不仅是创面愈合起始阶段的关键分子，并且在体内外都可诱导强有力的血管新生。我们前期初步临床研究中发现，糖尿病足溃疡组织中CD100表达较急性创面显著降低，但CD100表达下降是否和糖尿病足患者病情相关，补充外源CD100能否通过促进血管生成而促进创面愈合还不清楚。本课题拟在前期基础上扩大糖尿病足患者样本的收集，分析CD100及其受体表达同病情、病程及预后的关系；利用糖尿病小鼠慢性创面模型及缺氧高糖内皮细胞培养模型，通过加入外源CD100及调节受体信号，探讨其对创面愈合进程、血管生成作用及内皮细胞功能的影响，目的是阐明CD100参与糖尿病慢性创面愈合的作用及机制，为糖尿病慢性创面药物治疗探索新靶点。

糖尿病慢性创面是外科经常遇到的难题，目前在临床上仍缺乏有效的治疗方法。免疫信号蛋白CD100不仅是创面愈合起始阶段的关键分子，并且在体内外都可诱导强有力的血管新生。但是关于CD100和慢性创面愈合的关系还未有深入研究。方法 本研究选取人急性创面、慢性创面、及正常组织标本，用免疫组化染色的方法分析CD100分子在上述不同类型皮肤组织中的表达差异；制备BKS糖尿病小鼠创面模型，创面局部皮下注射CD100 250 ng（50µl），对照组注射PBS，术后隔天拍照计算创面面积占原始面积比率。HE和Masson染色作组织学检查，CD34免疫组化染色标记新生血管，CD68染色标记巨噬细胞检测评价炎症状态；CD100-/-小鼠制备创面模型， 通过外源补充CD100研究创面愈合的过程。.结果 人正常皮肤组织中CD100主要表达于表皮，其中胞膜高表达，真皮不表达；急性创面中表皮CD100膜表达水平显著降低。慢性创面中CD100在表皮组织低表达而真皮表达显著升高。糖尿病小鼠组织学结果表明第7、13、21天CD100组表皮与真皮的新生评分显著优于PBS组（P＜0.05）；肉芽组织厚度评分在第7、13天大于PBS组，第21天小于PBS组（P＜0.05），胶原重塑优于PBS组。在各个时间点CD100组血管化程度优于PBS组（P＜0.05）；炎症状态轻于PBS组（P＜0.05）。CD100-/-小鼠在创面建立后第13天，创面基本完全愈合，整个创面愈合过程中，补充CD100组小鼠创面愈合速度明显快于对照组，两组间差异具有统计学意义（P＜0.05）.结论 在人皮肤组织损伤早期，表皮组织膜型CD100的脱落可能对创面愈合发挥重要作用，慢性创面中表皮膜型CD100水平过低不能脱落释放到创面局部可能和创面难愈有关。在糖尿病小鼠和基因敲除鼠CD100-/-小鼠创面模型上，局部应用外源性的CD100分子，能够通过促进创面的血管化，减轻炎症反应促进糖尿病小鼠创面愈合。本项目研究为阐明CD100参与创面愈合过程的作用提供了理论及实验依据。可溶型CD100分子可能成为潜在的糖尿病慢性创面治疗药物。