经皮下注射携带siRNA的脂质微泡抑制乳腺癌淋巴管新生的实验研究

Breast cancer is a common malignancy in female. It is prone to have lymph node metastasis in early stage. Currently, several different treatment methods, such as surgery, radiotherapy and adjuvant chemotherapy are the effective treatment for the breast cancer. However, there are still some drawbacks for the treatment of lymph node metastasis. Lymphangiogenesis is important factor for inducing lymphatic metastasis. Ultrasound cavitation is one of the effective mechanisms for the treatment of lymph node metastasis. In our preliminary experiments, we injected liposome microbubbles subcutaneously to observe breast sentinel lymph node. In this study, by subcutaneous injection ultrasounic liposome microbubble carrying biological probes (siRNA) into breast cancer tumor edge, under low mechanical index(MI) mode, to observe lymphatic enhancement; Under high MI mode, by ultrasonic cavitation to release siRNA targeted towards to inhibit breast cancer lymphangiogenesis, preventing and treating breast cancer lymphatic metastasis. Uultrasound microbubble contrast agents have good stability under low MI mode, and provide the possibility to observe the status of lymphatic enhancement real time. This new type of molecule ultrasonic treatment has unique advantages with non-invasive, well-targeted, observation enhanced lymphatic in vivo. Our research content: to explore the mechanism of this method, to optimize the therapeutic effect, to observe incidental complications. The aim of this study is to explore new methods in prevention and treatment of lymph node metastasis in patients with breast cancer.

乳腺癌是女性常见的恶性肿瘤，早期易发生淋巴转移。目前有效的治疗方法手术、放疗、新辅助化疗在针对淋巴转移方面存在一定的缺陷。本研究建立在课题组经皮下注射超声造影剂检查乳腺癌前哨淋巴结的预实验基础上，以淋巴管新生是肿瘤淋巴转移的重要因素为理论基础，超声空化效应为机制，希望通过经乳腺癌肿瘤边缘皮下注射携带微血管/微淋巴管干扰基因(siRNA)的脂质微泡,低机械指数模式下观察淋巴管和淋巴结增强、高机械指数超声激发微泡空化的方法靶向释放siRNA以抑制乳腺癌淋巴管新生以预防和治疗乳腺癌淋巴转移。研究内容包括探讨该方法作用机制、优化治疗效果、观察可能的并发症等。脂质微泡在低机械指数模式下稳定性好、可实时观察淋巴管增强情况，这种新型的分子超声治疗方法具有非创伤性、良好的靶向性、具有活体显示淋巴管增强的独特优势。研究目的是探索一种乳腺癌淋巴转移预防和治疗的新方法。

VEGF是乳腺癌淋巴管新生的重要分子标志物，本课题通过超声空化机制爆破携带VEGFC siRNA的脂质微泡抑制乳腺癌淋巴管新生。首先通过建立裸鼠乳腺癌淋巴转移模型，并进行超声造影研究，为进一步超声空化提供实验基础，其次通过纳米级阳离子脂质微泡的制备，为实验提供了稳定、粒径小并携带阳离子的微泡，使其能够通过血管内皮细胞膜到达靶器官，并在此基础上，制备了携带VEGFC siRNA的靶向微泡，为靶向基因治疗乳腺癌淋巴转移提供了新方法.同时依据siＲNA序列设计合成以人VEGFCmＲNA全长序列为ＲNA的靶区设计3条VEGFC siＲNA序列，通过PCR、western blot等方法筛选siＲNA-C序列作为有效序列，其对VEGFC基因沉默效率最高，对乳腺癌细胞增殖抑制率最高，为乳腺癌超声空化机制治疗提供了新的靶点。超声空化机制爆破靶向微泡及si RNA-C序列对裸鼠乳腺癌淋巴转移模型中新生淋巴管抑制的研究仍在进行。综合上述结果，本研究为探讨VEGFC诱导乳腺癌淋巴管新生的研究奠定了坚实的实验基础。项目资助发表核心论文4篇。培养硕士研究生5名，其中3名已取得硕士学位，2名在读。项目投入经费49万元，支出 29.3897万元，各项支出基本与预算相符。剩余经费19.6103万元，剩余经费计划用于本项目研究后续支出。