诊断超声可视化控释载β-catenin-siRNA气泡对肝癌治疗及疗效监测一体化的实验研究

Hepatocellular Carcinoma (HCC)with high incidence, postoperative recurrence rate and mortality rate is one of the malignant tumors seriously threating to human life and health. Wnt is one of the key signaling pathways in the process of tumor occurrence and development, and β-catenin is the activation center in the Wnt signaling pathway.Researches show thatβ-catenin has a close contact with the development of liver cancer.If the expression ofβ-catenin protein is inhibited,the growth of liver cancer cells can be suppressed from several aspects,and even the tumor is destroyed. Ultrasound-targeted microbubble destruction (UTMD) can promote gene transfection and drug release so that it is widely used in the treatment study of disease. In traditional UTMD, ultrasonic instrument with low frequency ultrasound does not have the function of ultrasonic imaging, so it is very difficult to accurately position deep tumor of the body, which limit the further application of UTMD. Based on the successful preparation of nanoscale double carrier, anti-tumor researches in the project are carried out as follows: β-catenin-siRNA loading nano/micron bubbles are prepared.In-situ liver cancer model of naked rat is constructed,dignostic ultrasound is applied as a means of realtime controlled release.So the feasibility of the diagnostic ultrasound for implementing UTMD is evaluated and its applied parameters are optimized. And dignostic ultrasound with CEUS (contrast enhanced ultrasound)can quantity tumor blood perfusion. Application of dignostic ultrasound in UTMD can truly achieve the integration of the treatment of liver cancer, evaluation of therapeutic effect. This project explores the new treatment way for HCC.

原发性肝癌具有发病率高、术后易复发、死亡率高等特点，严重威胁人类生命健康。β-连环素（β-catenin）处于肿瘤发生、发展过程中关键信号传导通路Wnt的活化中心，研究表明β-catenin与肝癌发生发展的多个环节密切相关，如从抑制β-catenin蛋白表达入手，可从多方面抑制肝癌细胞生长。超声介导微泡破坏（UTMD）可促进基因转染用于疾病治疗。传统UTMD中治疗超声仪不具有超声成像功能，无法实现活体内深部脏器肿瘤的精准定位，使UTMD的应用受到限制。本项目在前期成功制备双载体纳米泡抗肿瘤研究的基础上，以裸大鼠肝癌原位种植瘤为研究对象，静脉注射载β-catenin-siRNA的纳米级/微米级气泡，以诊断超声辐照作为实时控释、爆破手段，并通过超声造影模式下肿瘤血流灌注评价肿瘤治疗效果。诊断超声应用于UTMD可以真正意义上实现肝癌治疗、疗效评估一体化，为肝癌的治疗提供新方法。

治疗性超声靶向破坏微泡应用于疾病治疗的效果显著，对于浅表部位病变易于定位及操作，对于活体内深部脏器肿瘤的治疗肿瘤实时定位是关键，而治疗性超声不具备成像功能。诊断性超声可清晰定位肿瘤，且通过调节超声功率可击破微泡，诊断性超声辐照微泡能实现动物活体内深部肿瘤治疗，真正实现实时超声控释的肿瘤特定部位药物、基因的定点释放。本项目拟制备稳定的载β-catenin-siRNA 的纳米级/微米级气泡载体，以裸鼠肝癌原位种植瘤为研究对象，以诊断用超声辐照作为实时控释、激发手段，观察诊断性超声介导 UTMD 治疗活体内肝癌的可行性。本项目制备了载siRNA的纳米泡，具有良好的超声造影显像效果，在治疗能量超声辐照下可爆破，从而释放siRNA。细胞毒性实验及溶血实验结果显示该载体具有良好的生物安全性，可以用于动物体内。体外细胞转染实验观察诊断用超声可实现细胞转染，达到较好的递送siRNA效果。比较两种方法建立裸鼠原位肝脏肿瘤模型，经腹壁切开直视下细胞液注射有更好的成瘤效果。超声辐照载β-catenin-siRNA/纳米泡抑制裸鼠肝脏原位肿瘤的研究，结果显示诊断超声可清晰显示肝内肿瘤所在位置，用于定位肿瘤，在造影剂聚集较多时进行辐照治疗，超声辐照载β-catenin-siRNA/纳米泡组较其他各组有很好的抑瘤效果，体积明显缩小，该组裸鼠生存期明显延长。诊断性超声可视化引导超声辐照裸鼠肝脏原位肿瘤实现深部脏器超声控释的β-catenin-siRNA/纳米泡的治疗。