HER2靶向载HSV-TK基因并具核定位效应的纳米超声造影剂构建及其在胃癌治疗中的应用基础研究

The herpes simplex virus thymidine kinase / ganciclovir (HSV-TK/GCV) system is one of the most promising suicide gene approach. Carrying the suicide gene into the tumor cell safely and effectively is the key. In this research, guanidination SS-PAAs (AGM-CBA) was prepared as the carrier for HSV-TK gene with effect of nucleation location; HER2 targeting nano-sized ultrasound contrast agent was prepared as the targeted carrier. After HSV-TK suicide gene was transfected into gastric cancer cells by ultrasound microbubble destruction technology (UTMD) and effect of nucleation location by AGM-CBA, the gastric cancer cells would then killed by the suicide gene with direct killing and bystander effect. The physical and chemical properties of the constructed HER2 targeted HSV-TK gene nano ultrasound contrast agent were investigated and finally optimized. Then, its anti-tumor effect on HER2 positive gastric cancer was evaluated by experiments in vitro and vivo in nude mice; and the effect of suicide gene transfection was also evaluated. Therefore, this research provide a new strategy for gastric cancer treatment by constructing HER2-targeted nano-sized ultrasound contrast agent carrying HSV-TK gene.

单纯疱疹病毒胸苷激酶/更昔洛韦（HSV-TK/GCV）治疗系统是目前最成熟的肿瘤自杀基因疗法，如何安全高效地将自杀基因靶向运输至肿瘤细胞内仍是函待解决的科学问题。在课题组研究基因载体的基础上，采用前期构建的胍基化SS-PAAs类聚合物（AGM-CBA）作为自杀基因的核定位载体，再利用HER2靶向纳米超声造影剂包裹作为基因的靶向载体。借助超声靶向微泡破坏技术和AGM-CBA载体的核定位效应共同提高自杀基因在胃癌细胞内的转染率，最终利用自杀基因的特性功能和旁观者效应杀灭胃癌细胞。对构建的HER2靶向载HSV-TK基因纳米超声造影剂进行理化性质等考察，并优化构建；通过体外实验和裸鼠移植瘤体内实验评价其对HER2阳性胃癌的抑瘤作用，并评估自杀基因的转染效果。本项目通过构建HER2靶向载HSV-TK基因纳米超声造影剂为HER2阳性胃癌的治疗策略提供新思路，并为其疗效及应用风险提供理论支撑与实验依据。

我国是胃癌高发国家，其中13%-16%为Her2基因扩增和蛋白质过表达。基于靶向精准治疗策略的发展，本课题组研究利用超声造影剂与自杀基因疗法结合，通过构建Her2靶向载HSV-TK自杀基因的纳米级超声造影剂，利用UTMD 技术提高HSV-TK自杀基因在胃癌细胞内的转染，为Her2阳性胃癌的治疗提供新思路。.在项目执行中，成功制备出HER2靶向载HSV-TK基因并具核定位效应的纳米超声造影剂（HCPAN），粒径约为441.1±128.3 nm，PDI为0.249，Zeta电位为28.1±5.26 Mv。透射电镜下单个纳米粒显示呈球形结构，形态规则。琼脂糖凝胶电泳显示，制得的HCPAN具有良好的抗核酸酶能力。酶标仪测得5×107个HCN纳米粒约能结合3μg HSV-TK质粒。体外实验HCPAN显示出优良的靶向性、转染效率及安全性：流式细胞仪检测其与细胞的结合率达87.8%；激光共聚焦显微镜观察证实其转染能力；MTT测得HCPAN无明显细胞毒性。联合LIFU对胃癌细胞的杀伤作用显著，并探索出合适的LIFU条件（2W/cm2，3min，1s on/1s off）。本研究显示的结果提示，将AGM-CBA用于保护HSV-TK基因，能增强HSV-TK基因的稳定性，且由于其具有一定的核定位效应，提高了基因的转染效率，为胃癌的治疗提供了新的技术方案。