TyrRGD靶向的GNPs-VEGFsiRNA增强肝癌RFA损伤效应的实验研究

Insufficiency damage lesion (residual tumor)is the main cause of recurrence of hepatocellular carcinoma after radiofrequency ablation(RFA) treatments, and enhancement of the damaging effects of RFA which expands the one-time damage volume is the key point to reduce residual tumor. Increasing the ion concentration within the foci and suppressing the infiltration and metastasis ability of residual cancer cells in the transition zone are important means to strengthen the damage effects of RFA. Gold nanoparticles are proved be non-viral vectors of gene targeting diagnosis and treatment and also as charged particles that posses superb thermal conductivity by shocking. Preliminary experiments show that: the topical application of nano gold solution can significantly increase the one-time damage volume of RFA, accompanied by a disadvantages of uneven distribution. Furthermore, These particles have no influence on biological behavior of residual tumor cells in transition zone. The significantly expression of integrin αvβ3 and VEGF of hepatoma cells and neovessels endothelial cells in cancer tissue have been confirmed，and that promote obviously infiltration and metastasis characteristics of residual tumor cells in the transition zone. Accordingly, this project intends to integratedly utilize the tissue characteristics of HCC, Principles of RFA, characteristics of Gold nanoparticles, Applying of cyclic pentapeptide RGD that can be closely integrated with αvβ3 as targeting molecules to build Gold nanoparticles complexes contained the cyclic pentapeptide RGD and VEGFsiRNA, thus to increase the targeted ion concentration within the foci which are evenly distributed, to suppress the infiltration and metastasis ability of residual cancer cells in the transition zone, using modern molecular biological techniques and the experiment in vivo and in vitro. To provide new methods for improving therapeutic effect of RFA for hepatocellular carcinoma.

病灶毁损不全（残癌）是射频消融（RFA）治疗肝癌后肿瘤复发的主要原因，增强RFA的损伤效应是减少残癌的重要方法，增加癌灶内离子浓度、抑制过渡区残癌细胞的侵润转移特性是增强RFA损伤效应的重要手段。已证明纳米金不仅是基因靶向诊疗的载体，也是导热性能极好的带电粒子。我们预实验表明：局部灌注纳米金溶液可明显增大一次性毁损体积，但分布不匀；对过渡区残癌细胞生物学行为也无影响。已证实肝癌细胞及癌组织内新生血管内皮细胞高表达整合素αvβ3受体，VEGF对过渡区残癌细胞侵润转移特性的增强起重要作用。因此，本课题拟充分整合利用肝癌组织特点、RFA原理、纳米金特性，应用可与αvβ3紧密结合的环五肽RGD做靶向分子，构建带有VEGFsiRNA的纳米金复合物；利用现代分子生物学技术及体内外实验手段，探索此复合物靶向均匀增加病灶内离子浓度、抑制过渡区残癌细胞侵润转移特性的作用。为提高肝癌RFA疗效提供新方法。

背景：病灶毁损不全（残癌）是射频消融（RFA）治疗肝癌后肿瘤复发的主要原因，增强RFA的损伤效应是减少残癌的重要方法，增加癌灶内离子浓度、抑制过渡区残癌细胞的侵润转移特性是增强RFA损伤效应的重要手段。已证明纳米金不仅是基因靶向诊疗的载体，也是导热性能极好的带电粒子。我们预实验表明：局部灌注纳米金溶液可明显增大一次性毁损体积，但分布不匀。对过渡区残癌细胞生物学行为也无影响。已证实肝癌细胞及癌组织内新生血管内皮细胞高表达整合素αvβ3受体，VEGF对过渡区残癌细胞侵润转移特性的增强起重要作用。因此，本课题拟充分整合利用肝癌组织特点、RFA原理、纳米金特性，应用可与αvβ3紧密结合的环五肽RGD做靶向分子，构建带有VEGFsiRNA的纳米金复合物。利用现代分子生物学技术及体内外实验手段，探索此复合物靶向均匀增加病灶内离子浓度、抑制过渡区残癌细胞侵润转移特性的作用，为提高肝癌RFA疗效提供新方法。.研究内容：1、TyrRGD-GNPs-VEGFsiRNA复合物的构建与鉴定. 2、TyrRGD-GNPs-VEGFsiRNA复合物增强RFA损伤效应的体外细胞实验研究. 3、TyrRGD-GNPs-VEGFsiRNA复合物增强RFA损伤效应的体内动物实验研究.重要结果：TyrRGD-GNPs-VEGFsiRNA复合物可明显增强RFA损伤效应，减少了RFA后残癌的发生，明显抑制了过渡区残癌细胞增殖、侵润转移，延长了荷瘤动物的生 存时间。.关键数据及其科学意义：单个肿瘤细胞中TyrRGD-GNPs-VEGFsiRNA颗粒数（29.2±4个）显著多于GNPs颗粒数（3.9±0.5个）；TyrRGD-GNPs-VEGFsiRNA组的RFA治疗毁损体积（5.12±2.12cm3）显著大于GNPs组（1.78±0.46cm3）和生理盐水组（1.49±0.44cm3）（P＜0.01）；TyrRGD-GNPs-VEGFsiRNA复合物组的肿瘤细胞凋亡（111.7±12.2）较GNPs组（36.3±3.8个）和生理盐水组（34.7±6.1个）明显增多（P＜0.01）。RFA联合TyrRGD-GNPs-VEGFsiRNA治疗组兔子的平均生存时间（70.9天）显著长于RFA联合生理盐水组（51.2天）及生理盐水组（43.9天）（P＜0.01）