纳米铁连接CD34×AFP双特异抗体介导内皮祖细胞携带内皮抑素/自杀融合基因治疗肝癌的研究

Endothelial progenitor cell (EPC) have the ability of targeted migrate and incorporate into sites of tumor neovascularization and participate in the development of tumor vascularization. Bispecific antibody (BsAb) might bind specifically the two types of cells. Superparamagnetic iron oxide nanoparticles (SPIO) were marked with In Vivo Tracking transplant EPC. This study intends to build of CD34 × AFP bispecific antibody which is anti-CD34 × anti-AFP and prepare complexes of SPIO-anti-CD34 × anti-AFP by connecting the SPIO with the BsAb. In the study, we plan to construct recombine-adenovirus by fusion gene combined endostatin gene with suicide gene, pAd-CMV-ES/CD, which were transfected into EPC in vitro. The EPC were labeled by the SPIO-anti-CD34 × anti-AFP whereafter and administrated into the rat hepatocarcinoma models via vascular. The implanted labeled EPC might be efficiently transfered into the hepatocarcinoma tissue duo to the anti-AFP immune oriented role and its capability of targeted migrating and incorporating into tumor neovascularization, and play dual role of fusion gene inhibition of angiogenic and direct anti-tumor cell; simultaneously the hepatocarcinoma tissue-targeted homing process of intravenously implantated EPC was monitored by MR in vivo. It might provide a new idea and method of immune-oriented EPC anti-tumor gene therapy for liver cancer and other malignancies..

内皮祖细胞(EPC)可归巢肿瘤参与肿瘤血管生成。双特异抗体(BsAb)可将两种效应细胞特异结合。采用超顺磁性氧化铁纳米粒子(SPIO)标记有可能活体示踪移植的EPC。本研究拟构建CD34×AFP双特异抗体anti-CD34×anti-AFP，将SPIO与该BsAb连接，制备成SPIO-anti-CD34×anti-AFP复合物(SPIO-BsAb)。构建内皮抑素和自杀基因的融合基因重组腺病毒pAd-CMV-ES/CD，体外转染EPC，然后以SPIO-BsAb标记EPC，将该EPC移植入肝癌大鼠体内，利用anti-AFP的免疫导向作用及EPC的肿瘤归巢性双重介导使EPC高效迁移到肝癌组织，发挥融合基因抑制肿瘤血管生成及直接杀伤肿瘤细胞的双重作用；同时采用磁共振(MR)成像活体监测EPC向肝癌组织的归巢过程。这将有可能为肝癌及其它恶性肿瘤的免疫导向EPC抗肿瘤基因治疗提供新思路和新途径。

本研究目的为将超顺磁性Fe3O4纳米粒子（SPIO）与双特异抗体anti-CD34×anti-AFP（BsAb）结合形成SPIO-anti-CD34×anti-AFP复合物SPIO-BsAb，构建内皮抑素（Endostatin）和自杀基因（CD）的融合基因重组腺病毒pAd-CMV-ES/CD，体外转染内皮祖细胞（EPC），然后以SPIO-BsAb标记EPC，将标记后EPC移植入肝癌小鼠体内，观察利用免疫介导和EPC归巢性介导EPC进入肝癌组织的可行性，以及内皮抑素和自杀基因抑制肿瘤血管生成和直接杀伤肿瘤细胞的有效性；采用磁共振（MR）成像活体示踪移植的EPC。本项目内容均已按计划完成。以共沉淀法制备纳米Fe3O4，化学偶联法制备CD34×AFP双特异抗体, 化学交联法制备成SPIO-anti-CD34×anti-AFP复合物。采集大鼠外周血，获取EPC细胞，进行分离、培养、鉴定，用RT-PCR 法扩增了CD基因和鼠源ES基因片段，用细菌内同源重组方法与腺病毒骨架质粒重组构建出重组腺病毒质粒pAd-CMV-ES/CD，并进行扩增和鉴定，获得了pAd-CMV-ES/CD体外高效转染EPC的最佳条件。采用共孵育法以SPIO-BsAb标记ES/CD融合基因修饰的EPC（ES/CD-EPC）,筛选出SPIO-BsAb标记EPC的最佳浓度。同时成功进行了标记细胞的体外MR成像。采用AFP阳性肝癌细胞株MHCC-97-H细胞接种至Balb/c裸鼠的右侧颈背部皮下，获得实体肿瘤，然后将肿瘤组织植入裸鼠肝叶实质内，以MR检测移植瘤的生长情况，获得稳定的AFP阳性肝癌动物模型。将SPIO-BsAb-ES/CD-EPC经尾静脉注入肝癌裸鼠模型体内，以MR成像在不同时间点动态、活体示踪了移植入裸鼠肝癌模型的细胞，将MR成像与肝脏肿瘤病理学、免疫组化、分子生物学分析等多种方法对照，发现SPIO-BsAb-ES/CD-EPC具有肝癌靶向性，能够抑制肿瘤血管生成和直接杀伤肿瘤细胞；MR成像可以活体动态示踪SPIO-BsAb-ES/CD-EPC，可能成为评价ES/CD-EPC靶向治疗肝癌疗效的重要新方法。本研究为肝癌及其它恶性肿瘤的基因治疗提供了新思路和新途径，并为肿瘤基因治疗提供了一种崭新的活体监测手段。本项目已发表SCI收录论文9篇，国内核心期刊收录论文1篇。