基于磁性荧光多功能探针USPIO- Cy5.5双模态成像精确清扫胃癌淋巴结研究

It is a long-sought goal for human to achieve the accurate dissection of metastatic lymph nodes in patients with gastric carcinoma. However, the key problem is how to pre-operatively differentiate benign from malignant lymph nodes, and how to intra-operatively locate these ones on a node-by- node basis accurately. In this project, we plan to synthesis a dual modality imaging probe with the lymphtropic ultrasmall superparamagnetic iron oxides (USPIO) conjugated with a biological NIR molecule dye (Cy5.5). The model of tumor-bearing lymph node in nude mice with gastric carciroma will be established to pre-operatively evaluate the efficacy of the probe in the diagnosis of lymph nodes by USPIO-Cy5.5 enhanced magnetic resonance imaging (USPIO-Cy5.5-MRI). Meanwhile, the power of differentiating benign from malignant lymph nodes in real-time will be also intra-operatively evaluate by near infrared fluorescence imaging. We will assess the effective window time of magnetic resonance and near infrared fluorescence imaging with USPIO-Cy5.5 in the diagnosis of lymph nodes in gastric carciroma. This study will provide the scientific and technical basis for the accurate dissection of metastatic lymph nodes in patients with gastric carcinoma.

努力开展淋巴结的精确清扫，实施胃癌最优个体化手术治疗一直是人们长期追求的目标，而能否术前准确地判断胃癌淋巴结转移，并在术中给予"点对点"(node-by-node) 的精确定位是实现这一目标的关键。本研究采用生物荧光染料Cy5.5偶联对淋巴结具有靶向亲和力的USPIO构建磁性荧光多功能纳米探针USPIO-Cy5.5。以裸鼠胃癌淋巴结转移模型为研究对象，通过USPIO-Cy5.5增强后的MRI和荧光成像，明确USPIO-Cy5.5-MRI术前诊断胃癌淋巴结的效能；评估术中USPIO-Cy5.5荧光实时成像区分转移和正常淋巴结的可靠性；摸索探针对淋巴结的有效评估时间窗口，为术前、术中准确判断胃癌转移淋巴结，实现胃癌最优个体化精确手术治疗提供技术支撑。

努力开展淋巴结的精确清扫，实施胃癌最优个体化手术治疗一直是人们长期追求的目标，而能否术前准确地判断胃癌淋巴结转移，并在术中给予“点对点”的精确定位是实现这一目标的关键。现有的常规MR单一成像方式由于不具备在术中实时成像淋巴结的功能，术前的影像学检查只能提供转移淋巴结的大概位置，手术医生难以在术中有效地将其与周围正常淋巴结相区分。本项目针对这一难题，.采用材料设计，化学合成的方法，制备出具有近红外成像功能的磁性氧化铁纳米探针，分析其体内外理化性质，并以裸鼠胃癌淋巴结转移模型为研究对象，对探针术前MR和术中荧光成像定位和诊断胃癌淋巴结的效能进行研究。.1、MR/NIR双模态探针的制备和理化性质研究：（1）通过偶联反应，使氨基化的超顺磁性的氧化铁表面接枝上氨基官能团。经分离纯化后，将染料Cy5.5经N-羟甲基琥珀酰亚胺（NHS）活化后修饰到氨基化的超顺磁性的氧化铁表面，获得了同时具有MR成像和近红外成像功能的磁性荧光探针。（2）在核磁共振仪上测定其弛豫率r2=0.9987mm-1s-1。通过绘制探针荧光强度衰减曲线，获得荧光的峰值时间30-45分钟，显影时间180-240分钟。（3）通过正常裸鼠体内测试，确定探针使用剂量为2.5mg/kg体重。.2、裸鼠胃癌淋巴结转移模型MR/NIR双模态探针成像研究：（1）通过裸鼠胃癌淋巴结转移模型的探针注射前后的MR成像，淋巴结信号强度未发生改变的淋巴结为转移淋巴结；信号下降的淋巴结为正常淋巴结。与病理结果对照，诊断的敏感性为95%、特异性为87%。（2）通过测量和分析探针增强后荧光图像上模型鼠胃周淋巴结的荧光强度，并记录相应位置。将高荧光数值的淋巴结视为为正常淋巴结；将低荧光数值和接近本底荧光水平的淋巴结视为为肿瘤转移淋巴结，并与传统的美蓝淋巴造影，证实了MR所显示的淋巴结与荧光成像显示的淋巴结为同一淋巴结。依据ROC曲线，明确有效区分良、恶性淋巴结的荧光强度阈值。这种兼具磁共振和近红外光学成像的双模态探针有望在胃癌转移淋巴结的精确清扫中提供技术支撑。