超声辐照联合微泡生物学效应对肿瘤局部化疗微环境影响及其机制的实验研究

Achieving optimal anti-tumor effects and alleviating the severe toxic side effects is the target during clinical tumor chemotherapy. Nowdays locally intratumoral injection of antitumor agents is the hot spot for tumor treatment. We have successfully made slow-released carboplatin microspheres with an optimization method,and injected them into many tumor models under the guidance of diagnostic ultrasound to see the treatment results.The result is encouraging that it works against the tumor that the tumor size grows slower compared with control group,but the tumor sizes still growed，and tumor cells didn't apoptosis in situ completely. Above all, we selected rabbit transplanted liver tumor as the research object, microbubbles enhanced ultrasound is irradiated on the tumor area after slow-released carboplatin microspheres are injected into the transplanted tumor. The diagnostic ultrasound with high mechanical index was used in microbubble destruction to enhances tumor cell membrane permeability,meanwhile the treatment conditions were optimized to improve the therapeutic effect and reduce the adverse effect.In order to clarify the detailed mechanism, Lanthanum nitrate tracer transmission electron microscopic was used to observed cell membrane permeability. In addition, the tumor microenvironment factors were investigated to clarify the mechanism for the ultrasound sonication with microbubbles on the biological effect of intratumor chemotherapy treatment.The project is designed to develop the original local injection of slow-released carboplatin microspheres treatment system,and lay the theoretical foundation for the interventional tumor treatment guided by ultrasound strategy.

既疗效好又无严重毒副作用是肿瘤化疗的目标。瘤内注射仍是当今治疗恶性肿瘤的研究热点。申请人前期以优选法成功制备卡铂缓释微球，超声引导经皮穿刺瘤内注射对多个肿瘤模型进行了探索性治疗，取得了很好疗效，但未达到完全原位灭活肿瘤细胞目的。本课题以兔肝移植瘤为研究对象，采用超声引导瘤内注射卡铂缓释微球同时静脉注射超声微泡，并对肿瘤区进行超声辐照。利用高机械指数诊断超声爆破微泡提高肿瘤细胞膜通透性，以期提高卡铂的生物利用率，达到最佳治疗效果、降低毒副作用之目的；并通过硝酸镧示踪法透射电镜证明肿瘤细胞及血管内皮细胞膜通透性改变。探寻肿瘤组织生长微环境相关因子改变，以阐明超声辐照联合微泡生物学效应对化疗缓释微球瘤内注射治疗肿瘤内环境改变影响及其作用机制。该项目是对原有卡铂缓释微球肿瘤瘤内注射治疗系统研究的改进与发展，为进一步完善介入超声治疗恶性肿瘤新方法提供理论依据。

背景：.本实验拟以兔VX2移植瘤及VX2体外培养细胞为研究对象，采用超声引导瘤内注射卡铂缓释微球同时静脉注射微泡造影剂，并对肿瘤区进行超声辐照。利用超声辐照微泡爆破产生生物学效应以提高肿瘤细胞膜通透性，以期提高卡铂的生物利用率，达到最佳治疗效果、降低毒副作用之目的。.研究内容：.优化制备卡铂缓释微球。对移植瘤行HE及Tunel染色，分析肿瘤凋亡指数。体外培养VX2细胞进行细胞cck8比色，流式细胞术探讨各组肿瘤细胞凋亡情况。透射电镜观察肿瘤细胞膜及血管内皮细胞膜通透性改变。使用DH-2010A远红外热像仪观察肿瘤区域温度情况。以IHC、RT-PCR、WB检测肿瘤相关生长因子Hif-1α、ICam-1表达情况；同时对不同超声联合微泡辐照体外卡铂缓释微球释药情况进行观察。.结果与意义：.制作出粒径5-100μm，包封率74.8%，载药率约10-13%的卡铂缓释微球，使其更容易输送到达靶向器官或结构组织。各组凋亡指数：瘤内注射卡铂缓释微球+微泡造影剂+诊断超声辐照肿瘤组14.9±1.59%，单纯瘤内注射卡铂缓释微球组10.8±2.13%、超声联合微泡辐照组5.16±0.42%、对照组3.0±0.63%。卡铂缓释微球组细胞膜连续性尚可，细胞膜稍欠光整。S2000彩色超声诊断仪联合微泡+卡铂缓释微球组、超声治疗仪联合微泡+卡铂缓释微球组肿瘤细胞高尔基体、线粒体呈水肿状，细胞膜不均匀改变，部分区域变薄、连续性差、可见小孔隙现象。通过IHC、RT-PCR、WB等方式检测各组肿瘤组织肿瘤相关因子Hif-1α，其受超声联合微泡造影剂辐照及微球释放化疗药物影响呈持续性减低表达，而细胞内Icam-1呈增高改变，考虑为细胞损伤后炎性反应。AP-170超声联合微泡辐照卡铂缓释微球VX2肿瘤组织温度发生改变，可升高至40°C。体外释药实验证明温度升高可促进卡铂缓释微球化疗药物释放。体外培养VX2细胞实验证明，细胞持续受到卡铂缓释微球的作用，被持续诱导凋亡，较卡铂原药组有明显的持续抑制体外培养肿瘤细胞生长的效果。因此，超声联合微泡辐照卡铂缓释微球对VX2肿瘤化疗疗效有一定促进作用，为进一步完善介入超声治疗恶性肿瘤新方法提供理论依据。