清热化瘀方对肛瘘术后创面修复Src/VE-cadherin通路相关因子调控机理研究

Anal fistula operation is contaminated wound.It has significantly inflammatory exudation ,and the healing of the wounds was more difficult.Inflammatory response in wound after operation often because of damp-heat,in spite of which symptom it is before surgery.That red swelling,heat pain and exudation of wound is he symptom of damp-heat, and vascular permeability increased is the key of its modern pathogenesis.The degree of permeability relate to both inflammatory exudation and the wax and wane of damp-heat.The study of open wound around anus must pay attention to inflammatory and exudation.Most study of wound healing view to tissue repair at home and abroad,and there was less study on influence of exudation on wound healing.Based on Summary of the experiments, the studying team study on therapeutic mechanism of external washing method for clearing heat and eliminate dampness.They Primarily found that regulate vascular permeability is important link of Improve the syndrome of inflammatory exudation , damp-heat and promoting wound repair.Research of Foreign also display vascular permeability is important link of regulate inflammatory exudation and promoting wound repair,it is a important field. We from optical microscope ,laser confocal,mRNA and protein level angle to study the mechanism that external washing method for clearing heat and eliminate dampness regulate vascular permeability promoting inflammatory exudation though Src/VE-cadherin signal pathway.And explore the mechanism that Traditional Chinese Medicine maybe regulate vascular permeability control inflammatory to promote wound healing,and so as to find more Scientific and Effective external washing method of TCM.

肛瘘手术切口是开放污染伤口，炎症渗出明显，不易愈合，是临床难题。课题组经长期临床实践制定的清热化瘀方促进创面愈合疗效显著，但其作用机制尚不清楚。研究表明：术后创面红肿热痛渗出的表现，其现代医学的核心改变之一是血管通透性增加。通透性改变既和炎症渗出有关，又与创面愈合进程、质量密切关联。我们前期研究证实清热化瘀方能明显改善创面炎症渗出，初步探讨其机制可能通过调控血管通透性Src/VE-cadherin信号通路发挥治疗作用。在此基础上，本研究拟运用光镜、透射电镜研究Src/VE-cadherin信号通路调控血管内皮紧密连接的机制，阐释该方促进创面愈合的机理；并进一步观察信号通路相关因子VEGF、Ang1、Tie2表达、分布规律，从mRNA、蛋白及磷酸化表达等水平诠释该方调控血管通透性促进创面愈合的内在机制，以期发现中药复方治疗创面愈合的作用靶点，为难治性创面愈合提供新的思路和方法。

肛瘘手术切口是开放污染伤口，炎症渗出明显，不易愈合，是临床难题。课题组经长期临床实践制定的清热化瘀方促进创面愈合疗效显著，但其作用机制尚不清楚。我们前期研究证实清热化瘀方能明显改善创面炎症渗出，探讨其机制可能通过调控血管通透性Src/VE-cadherin信号通路发挥治疗作用。在此基础上，本研究运用光镜、透射电镜研究Src/VE-cadherin信号通路调控血管内皮紧密连接的机制，阐释该方促进创面愈合的机理；并观察信号通路相关因子VEGF、Ang1、Tie2表达、分布规律，从mRNA、蛋白及磷酸化表达等水平诠释该方调控血管通透性促进创面愈合的内在机制，以期发现中药复方治疗创面愈合的作用靶点，为难治性创面愈合提供新的思路和方法。本项目以SD大鼠肛旁创面模型模拟人类肛瘘创面为研究对象，在造模后第3、7、14天肉眼及电镜观察创面渗出、炎症及愈合情况，结果提示清热化瘀方能减轻创面炎症反应，较快的促进创面愈合，电镜下观察清热化瘀组胶原纤维分布致密、规则，纤维束排列紧密厚实，相邻纤维束之间空隙较小；光镜下HE染色清热化瘀方组新生毛细血管丰富且成纤维细胞排列整齐，Masson's trichrome三色染色法结果提示清热化瘀3号方组的胶原合成较为丰富，胶原层较厚，且排列致密有序；伊文氏蓝染色法提示清热化瘀1号方早期能降低血管通透性，清热化瘀3号方能增加血管通透性，清热化瘀方能调控血管通透性的改变以控制创面局部炎症程度；PCR结果显示清热化瘀1号方造模3天SRC基因mRNA表达量明显较低，而 VE-cadherin/VEGF基因mRNA表达量升高，造模后第7天基因表达量相反；免疫组化及免疫荧光提示造模后7天清热化瘀1号方、高锰酸钾 VE-cadherin蛋白的表达阳性明显，清热化瘀1号方能降低血管通透性，创面愈合早期应加重清热利湿药物的应；Western-Blot提示热化瘀1号方组src蛋白表达明显低于其他组，同时Ang1蛋白表达量明显高于其他组别，提示创面修复早期应用清热利湿药物能明显降低血管通透性，减轻局部炎症反应，以利于创面愈合。结果表明清热化瘀方有效地降低血管通透性，减少创面组织水肿，控制炎性渗出，促进肉芽组织胶原纤维的合成，提高创面愈合率；同时有效促进创面组织VE-cadherin 的基因和蛋白表达，这可能是清热化瘀方控制炎症，改善局部湿热症状，促进创面愈合的分子机制。