CLARITY技术构建树鼩肝癌血管三维成像及相关研究

Angiogenesis plays a key role in hepatocarcinogenesis and metastasis. At present, studies of angiogenesis are limited in two-dimensional level, so the changes of the vascular spatial structure are not able to be shown correctly and measured accurately. Moreover, information on the regulatory mechanism of angiogenesis is not able to be obtained directly from intact tissue either. A new technology named CLARITY solves these technical problems. CLARITY technology creates an intact tissue that is permeable to both visible-spectrum photons and exogenous macromolecules and preserves genetic information. After immunofluorescence, interesting biologic information is formed to three-dimensional imaging by confocal microscopy. In our study, some tree shrews are induced hepatocarcinogenesis by feeding aflatoxin B1. This tree shrew's livers are made transparent and permeable by CLARITY technology, and then are marked CD34 and VEGF by immunofluorescence. Finally Angiogenesis in HCC is formed to three-dimensional imaging by confocal microscopy. The three-dimensional changes of angiogenesis will be learned and measured accurately, the relation between angiogenesis and regulating factor will be investigated also. It will provide a new clue for treatment targets to cure hepatocellular carcinoma.

新生血管形成在肝癌发生发展以及转移过程中起着关键作用，而目前肝癌血管研究局限在二维层面，无法体现肝癌血管形成过程空间结构变化及精确测量，此外肝癌血管调控机制等信息也无法直接从完整组织中获取。CLARITY技术解决了这些技术难题。CLARITY技术构建可渗透大分子并保留生物遗传信息的透明完整组织模型，免疫荧光标记后运用激光共聚焦显微镜实现研究对象三维成像，为肝癌血管形成研究提供了一个全新的技术手段。本研究以树鼩为研究对象，运用CLARITY技术构建黄曲霉毒素B1诱导的树鼩肝癌形成各个阶段肝脏透明模型，免疫荧光标记血管内皮细胞CD34和血管生成因子VEGF后，运用激光共聚焦显微镜三维成像。了解肝癌血管形成过程三维结构变化并量化及与血管生成调控因子的相互关系，为肝癌治疗靶点提供新的线索。

本项目通过CLARITY技术构建DEN诱导大鼠肝癌发生各个阶段肝脏透明模型，运用血管SMA和4型胶原蛋白抗体（collagen-IV）进行肝脏血管荧光染色后，运用激光共聚焦显微镜扫描并计算机合成三维图像，从而了解并量化肝癌血管三维结构变化。获得项目资助以来，本课题组按照年度计划开展研究，虽然做了部分调整，总体上完成了各项研究任务，取得以下成果：（1）优化CLARITY技术构建DEN诱导大鼠肝癌模型肝脏透明化模型条件。（2）优化肝脏透明化组织标本特异性荧光标记及激光共聚焦扫描计算机成像条件。（3）CLARITY技术在病理石蜡切片延伸运用，并申报国家专利一项。综上所述，CLARITY组织透明化技术运用为肿瘤发生发展的生物微观结构研究提供技术支持。