基于肠系膜淋巴管荧光动态造影技术的分子探针HA-ICG判定克罗恩病肠道手术切缘的精准治疗研究

It is a difficult problem that there is no such an objective criteria for judging the margin of resection during intestinal surgery and high recurrence rate in anastomotic area of postoperative intestinal inflammation in patients with Crohn's disease. The pathological changes of lymphatic vessels are not only closely related to the inflammation of the intestinal wall, but also may be the key to reduce the recurrence rate of anastomotic site. This project intends to target mesenteric lymphatic vessel as the observation point, produce fluorescent molecular contrast agent, a chemical compound combined with hyaluronic acid and indocyanine green (HA-ICG), targeting the lymphatic endothelial cells, and conducting dynamic fluorescence imaging on a rat model. With image postprocessing, the drainage function of the mesenteric lymphatic vessels were measured and analyzed. After confined with pathology immunohistochemical analysis, we could explore the following ideas: First, establishment of a kind of novel method of dynamic observation, measurement, evaluation of mesenteric lymphatic vessel drainage function dynamically and specifically. Second, analysis of the feasibility of evaluationn of intestinal mucosal inflammation activity by using extra-luminal changes of mesenteric lymphatic drainage function. Try to establish the imaging diagnostic method which could give a comprehensive assessment of both the intra or extra intestinal luminal lesions. All of which could create the theoretical foundation for establishment the criteria of margin of resection in Crohn's disease in the future.

克罗恩病肠道切除吻合手术缺乏客观的切缘判定标准及术后的吻合口区肠道炎症高复发率是一直以来困扰医学界的一个难题。目前的研究表明，淋巴管的病变不仅与克罗恩病肠壁炎症密切相关，也很可能是降低肠道手术吻合口炎症复发率的一个关键所在。本项目拟以肠系膜淋巴管作为观察靶点，制备靶向淋巴管内皮细胞的荧光分子造影剂透明质酸-吲哚菁绿化合物（HA-ICG），采用大鼠模型活体动态荧光成像技术，辅以图像后处理，对肠系膜淋巴管的引流功能进行测定，结合病理免疫组化分析，探索：一、建立一个能够活体动态观察、测量、评估肠系膜淋巴管引流功能的特异性的靶向成像方法。二、分析以肠腔外肠系膜淋巴管引流功能的变化来反应肠腔内粘膜炎症活动度的可行性，探索一种可综合评估肠腔内/外病变情况的影像诊断方法，为今后以此为依据建立克罗恩病肠道手术切缘判定标准打下理论基础。

克罗恩病肠道切除吻合手术缺乏客观的切缘判定标准及术后的肠道吻合口炎症高复发率的原因多年来一直困扰着致力于炎症性肠病研究的学者。既往的研究提示克罗恩病中肠壁淋巴管的病变不仅与肠壁炎症密切相关，也很可能是降低肠道手术吻合口炎症复发率的一个关键所在。本项目以肠系膜淋巴管作为观察靶点，制备了靶向淋巴管内皮细胞的荧光分子造影剂透明质酸-吲哚菁绿化合物（HA-ICG），利用大鼠硫酸葡聚糖（DSS）肠炎模型活体动态荧光成像技术并辅以图像后处理，对肠系膜淋巴管的引流功能及其与肠壁黏膜炎症活动的关联进行了探索，得到如下结果：一、研究中制备所得的HA-ICG体外理化性质稳定、无毒副作用，活体荧光靶向显影效果好；二、慢性肠炎组大鼠肠壁存在明显的微淋巴管增生，但荧光动态成像提示其系膜淋巴管回流速度最慢，这可能是基于微淋巴管内皮肌化导致的肠壁淋巴组织液单向回流障碍；三、HA-ICG在组织间质内的扩散速度较ICG变慢，但慢性肠炎大鼠的HA-ICG回流速率大于ICG，提示在慢性肠炎状态下，HA-ICG显示出更好的靶向性造影效果；四、模型大鼠肠壁微淋巴管肌化率与淋巴荧光动态造影回流速率之间存在负性线性关系，线性回归方程为y=-2.469x+2.953（p=0.025）。因此，本项研究建立了一个能够活体动态观察、测量、评估肠系膜淋巴管引流功能的荧光靶向成像方法，同时发现慢性肠炎状态下肠壁微淋巴管肌化可能是导致组织液回流障碍及炎症持续存在的原因，为今后以此为依据建立克罗恩病肠道手术切缘判定标准打下理论基础。