【作者】 杨健；

【导师】 吴小翎；

【作者基本信息】 重庆医科大学 ， 内科学， 2012， 博士

【摘要】 肝癌是世界范围内发病率和死亡率均较高的常见恶性肿瘤之一，2011年全球癌症统计数据显示我国肝癌的新发和死亡患者数量约占世界总数的二分之一。近年来，随着我国医疗技术和综合经济水平的不断提高，肝癌的手术治疗率逐渐提高，但是对于不适宜手术治疗或者复发转移的患者，采用包括靶向化疗、系统化疗在内的系统治疗方法对于延长患者的生存时间非常重要；传统的化疗方法存在靶向作用差、毒副作用较大、患者耐受差、综合疗效欠佳等不足导致临床应用受限，因此，迫切需要尽快研发出一种靶向性较高、安全性较好、抗肿瘤作用较强，且毒副作用较小的新型治疗方法。随着近年来国内外超声分子影像学技术的不断发展，靶向超声分子探针提供了一种靶向运载化疗药物（或基因）到达肿瘤局部组织、并在适当强度的超声作用下靶向释放化疗药物（或基因）的新型手段，有助于提高化疗药物的靶向性和抗肿瘤效果。死亡受体5的单克隆抗体（DR5mAb）可以特异性的与肝癌细胞表面表达升高的TRAIL-R2（即DR5）结合，并通过DR5传递细胞凋亡信号，进而诱使肝癌细胞发生凋亡，同时由于DR5在正常肝脏组织中低表达，DR5mAb不会引起正常肝细胞凋亡，所以DR5mAb可以作为肝癌靶向治疗的一种理想物质。多烯紫杉醇（docetaxel, DTX），又名多西他赛，是一种紫杉类抗肿瘤药物，其作用机制是在细胞周期中促进微管聚合并增强微管稳定性，且DTX拥有比紫杉醇更强的抗肿瘤活性；研究表明将DTX与超声造影技术结合可以改善DTX的溶解性，且对肝癌等肿瘤具有明显的抑制作用。因此，在本实验中，我们利用靶向超声分子探针制备技术和生物素-亲和素方法将DR5mAb与DTX药物微泡相连接，检测DR5-靶向载DTX脂质微泡（DR5-targeteddocetaxel-loaded lipid microbubbles, DTDLLM）的抗体连接情况和理化性质，研究靶向载药微泡与肝癌细胞的靶向结合能力，并通过细胞学实验和动物模型研究DTDLLM结合超声靶向微泡破裂技术对肝癌的作用及相关机制，从而为肝癌靶向治疗和超声分子影像技术研究提供新的实验依据。本课题主要包括以下三部分内容：第一部分DR5-靶向载DTX脂质超声微泡的制备、检测和体外寻靶能力实验目的制备一种携带DR5mAb的DTDLLM，检测其性质并探讨其特异性体外寻靶能力。方法采用机械振荡法制备载DTX的脂质超声微泡（DLLM）；制备生物素化的肝癌DR5mAb，通过生物素-亲和素连接法将DR5mAb连接于DLLM表面。采用免疫荧光法检测微泡与DR5mAb的连接情况，观测DTDLLM的稳定性，检测靶向微泡的粒径、浓度、zeta电位、载药量、包封率等性质，探讨其体外寻靶能力，并与非靶向的DLLM组进行比较。结果DTDLLM平均粒径1232nm，平均浓度为3.1×109/ml，微泡的包封率为73.5%，平均载药量为25.3%，免疫荧光法检测发现DTDLLM表面有绿色环状荧光分布，在体外寻靶实验中靶向微泡组HepG2细胞周围可见较多DTDLLM微泡紧密结合，而对照组肝癌细胞未发现特异性微泡结合。结论成功制备了携带肝癌DR5mAb的DTDLLM，该微泡能够与HepG2细胞牢固结合。DTDLLM有望成为一种新型、高效、可用于超声分子显像的肝癌靶向药物载体。第二部分DR5-靶向载DTX脂质超声微泡联合UTMD对肝癌HepG2细胞作用和机制的实验研究目的研究DTDLLM联合UTMD对肝癌细胞HepG2的增殖、细胞周期和细胞凋亡，以及Caspase-8、Bcl-2和DR5的mRNA和蛋白表达水平的影响。方法体外培养人肝癌细胞HepG2，实验分9组，分别是：空白对照组（Con），DTX组（DTX），DTX联合超声组（DTX+US），空白微泡组（MB），空白微泡联合超声组（MB+US），载DTX微泡组（DLLM），载DTX微泡联合超声组（DLLM+US），DR5-靶向载DTX微泡组（DTDLLM），DR5-靶向载DTX微泡联合超声组（DTDLLM+US）。CCK-8法检测各组细胞的增殖抑制情况，流式细胞术检测细胞周期情况，TUNEL法检测细胞凋亡情况，RT-PCR及Western Blot分别检测各组处理后的Caspase-8、Bcl-2和DR5的mRNA和蛋白表达情况。结果在9个实验组中，DR5-靶向药物微泡联合超声组对HepG2细胞的增殖抑制作用最强（P＜0.01），且有比较明显的时间和浓度效应关系，与其他处理组进行比较，DTDLLM+US组对HepG2细胞的凋亡诱导作用最强（P＜0.01），且将HepG2细胞周期阻滞于G2/M期，该组的Caspase-8和DR5的mRNA和蛋白表达水平明显增高（P＜0.01），且Bcl-2mRNA和蛋白表达水平明显降低（P＜0.01）。结论DTDLLM对肝癌细胞HepG2的增殖有明显抑制作用，且可将肝癌细胞周期阻滞于G2/M期，并诱导肝癌细胞发生凋亡，上述作用强于非靶向的DLLM以及DTX，其作用机制与影响凋亡相关的Caspase-8、Bcl-2和DR5的mRNA和蛋白表达水平有关。第三部分：DR5-靶向载DTX超声微泡联合UTMD对裸鼠肝癌移植瘤作用和机制的实验研究目的研究DTDLLM联合UTMD对裸鼠肝癌移植瘤的治疗作用和相关机制。方法建立裸鼠肝癌皮下移植瘤模型，实验分5组，分别是：空白对照组（Con），DTX组（DTX），DTX联合超声组（DTX+US），载DTX微泡联合超声组（DLLM+US），DR5-靶向载DTX微泡联合超声组（DTDLLM+US）。测量治疗前后各处理组肿瘤体积，绘制裸鼠肿瘤的生长曲线，并计算肿瘤抑制率，TUNEL法检测各处理组细胞的凋亡情况，免疫组织化学染色法检测肿瘤组织MMP-2和VEGF的表达，实时定量PCR及Western Blot分别检测各组Caspase-8、Bcl-2和DR5的mRNA和蛋白表达情况。结果各实验组相比较，DTDLLM+US组对裸鼠肝癌的抑制率最高，其凋亡指数明显高于其他组（P＜0.01），增殖指数明显降低（P＜0.01），MMP-2和VEGF的表达水平均明显低于其他组（P＜0.01），RT-PCR和WB结果显示：DTDLLM+US组Caspase-8和DR5的mRNA和蛋白表达水平均明显增高（P＜0.01），Bcl-2mRNA和蛋白表达水平明显低于其他处理组（P＜0.01）。结论DTDLLM对裸鼠肝癌的生长有明显抑制作用，且抑瘤作用强于非靶向的DLLM和DTX，作用机制与其能够更强的抑制裸鼠肿瘤微血管生成和浸润转移、上调Caspase-8和DR5表达水平、下调Bcl-2表达水平有关。DTDLLM联合UTMD具有比非靶向微泡以及单纯药物治疗更佳的肝癌靶向作用和更强的抗肿瘤作用，为肝癌的靶向治疗和靶向超声造影剂的分子影像治疗提供了依据。更多还原

【Abstract】 Liver cancer is one of the common worldwide malignant tumors withhigh morbidity and mortality, and global cancer statistics in the year2011showed that almost half of world’s newly diagnosed and death cases ofliver cancer were reported in China. In recent years, with the continuousimprovements of our medical technology and economic levels, the rates ofsurgical treatment of liver cancer have gradually increased, while on theother hand, multimodal therapy including chemotherapy could prolongpatients’ survival time and therefore plays an important role in patients withrecurrented or metastasized liver cancer. But traditional methods ofchemotherapy always result in poor targeting ability and efficacy, as well asside effects and lack of patient tolerance, and therefore there remains anurgent need to develop new drug delivery systems with improved targetingability and antitumor effect as well as alleviated side effects.With the constant developments of ultrasound molecular imaging technology in recent years, ultrasound-targeted molecular probes combinedwith ultrasound targeted microbubble destruction (UTMD) provide us anew method of noninvasive tumor-targeted drug delivery withcontrol-releasing ability, which may help us to improve the targeting abilityand antitumor efficacy of chemotherapeutic drugs for the treatment ofrecurrented liver cancer.Anti-death receptor5monoclonal antibody (DR5mAb) canspecifically bind to TRAIL-R2(death receptor5，DR5) which has elevatedexpressions on the surface of liver cancer cell, and then induce apoptosis inliver cancer cells through intracellular apoptotic pathways, however,DR5mAb would not cause hepatocyte apoptosis in normal liver tissuesbecause of their low expression of DR5, and therefore DR5mAb could beused as an ideal targeting material for the treatment of liver cancer withlittle damage in normal liver tissue.Docetaxel (DTX) is one promising antitumor drug of the taxane class,and plenty of researches have demonstrated that DTX has a more powerfulantitumor effect than paclitaxel. DTX could promote microtubulepolymerization and enhance microtubule stability in cell cycles, and recentstudies have demonstrated that lipid-coated docetaxel combined withUTMD could achieve effective improvements of docetaxel solubility andsignificant inhibition of liver cancer. Therefore, in this study, DR5mAbwere binded to DTX-loaded microbubbles via biotin–avidin technology, and then physical properties as well as targeting ability of microbubbleswere detected. Effect and mechanisms of DR5-targeted microbubblescombined with UTMD on liver cancer were investigated throughcytological experiments and animal models, which may provide newexperimental evidence for targeting treatment of liver cancer and futherresearch on ultrasound molecular imaging.The main subject of this study includes the following three parts:PARTⅠPREPARATION AND TARGETING ABILITY OFDR5-TARGETED DOCETAXEL-LOADED LIPIDMICROBUBBLES IN VITROObjective To develop a novel DR5-targeted and docetaxel-loadedmicrobubble and investigate its characterizations and specific targetingfunction in vitro.Methods DR5-targeted docetaxel-loaded lipid microbubbles(DTDLLM) were constructed by the avidin-biotin interaction which bindedbiotinylated DR5mAb to the surfaces of DLLM prepared by mechanicalvibration. The immunofluorescent assay was used to test the combinationbetween DR5mAb and DLLM. Microbubble stability was preliminarilyevaluated. The physical properties were determined and microbubblecomposition was optimized. Compared with DLLM as control, themorphology and targeting ability of DTDLLM to HepG2cells wereinvestigated in vitro. Results DTDLLM prepared in this study have similar particle sizewhich was1232nm on average, and were well dispersed with theconcentration of3.1×109/ml. The average drug entrapment efficiency anddrug-loaded amount was73.5%and25.3%, respectively. We also observedthat green fluorescents were obviously expressed around the surface ofDTDLLM, and targeted microbubbles tightly conjugated with HepG2cellsin DTDLLM group while few microbubble was observed binding to HepG2cells in DLLM group.Conclusions DTDLLM can be successfully constructed andspecifically attached to HepG2cells in vitro. DTDLLM，as a novel contrastagent with good properties and superior targeting ability, may become oneof the effectively targeting drug deliveries for ultrasound molecularimaging and HCC targeting therapy.PARTⅡ EFFIECT AND MECHANISMS OF DR5-TARGETEDDOCETAXEL-LOADED LIPID MICROBUBBLES COMBINEDWITH ULTRASOUND TARGETED MICROBUBBLEDESTRUCTION ON HEPG2CELLS IN VITROObjective To study the proliferation-inhibited and apoptosis-inducedeffects of DR5-targeted docetaxel-loaded lipid microbubbles (DTDLLM)combined with ultrasound targeted microbubble destruction on HepG2cellsin vitro, and investigate mRNA and protein expression levels of Caspase-8,Bcl-2and DR5. Methods Human hepatoma cell line HepG2were cultured and dividedinto nine groups, including the control group (Con), docetaxel group(DTX), docetaxel combined with ultrasound group(DTX+US), puremicrobubbles group(MB), pure microbubbles group combined withultrasound group(MB+US), docetaxel-loaded lipid microbubbles group(DLLM), docetaxel-loaded lipid microbubbles combined with ultrasoundgroup (DLLM+US), DR5-targeted docetaxel-loaded lipid microbubblesgroup (DTDLLM), DR5-targeted docetaxel-loaded lipid microbubblescombined with ultrasound group (DTDLLM+US). The CCK-8and TUNELassay were used to detect the proliferation-inhibited and apoptosis-inducedeffects, retrospectively, and then cell cycles were analyzed by flowcytometry. The mRNA and protein expression levels of Caspase-8, Bcl-2and DR5were tested by RT-PCR and Western Blot.Results Of all the groups, DTDLLM+US group demonstrates thestrongest proliferation-inhibited effect with an obvious time-dose-responserelationship (P<0.01), and the strongest apoptosis-induced effect (P<0.01)as well as an increased G2/M phase arrest showed by flow cytometrydetection of HepG2cell cycle (P<0.01). The mRNA and protein expressionof Caspase-8and DR5in DTDLLM+US group were significantlyup-regulated (P<0.01), and those of Bcl-2were conspicuouslydown-regulated (P <0.01) when compared to other groups.Conclusions DTDLLM combined with UTMD can significantly inhibit the proliferation of HepG2cells, and increase G2/M phase arrest aswell as apoptosis-induced effect, which resulted from regulated mRNA andprotein expressions of apoptosis-related factors including Caspase-8, DR5and Bcl-2.PART Ⅲ EFFIECT AND MECHANISMS OF DR5-TARGETEDDOCETAXEL-LOADED LIPID MICROBUBBLES COMBINEDWITH ULTRASOUND TARGETED MICROBUBBLEDESTRUCTION ON LIVER CANCER IN NUDE MICEObjective To investigate the antitumor effect and mechanisms ofDR5-targeted docetaxel-loaded lipid microbubbles (DTDLLM) on livercancer in nude mice.Methods Subcutaneous hepatoma was established in nude mice andthen randomly divided into five groups, including the control group (Con),docetaxel group(DTX), docetaxel combined with ultrasound group(DTX+US), docetaxel-loaded lipid microbubbles combined with ultrasoundgroup (DLLM+US), DR5-targeted docetaxel-loaded lipid microbubblescombined with ultrasound group (DTDLLM+US). Tumor growth curvewere drew, and tumor inhibition rate (TIR) were calculated. The TUNELassay was used to detect apoptosis-induced effects, and expression ofMMP-2and VEGF were detected by immunohistochemistry (IHC). ThemRNA and protein expression levels of Caspase-8, Bcl-2and DR5weretested by RT-PCR and Western Blot methods. Results Compared with other groups, DTDLLM+US demonstrates thehighest TIR (P<0.01) and AI (P<0.01) as well as the lowest expression ofMMP-2(P<0.01) and VEGF (P<0.01). The mRNA and protein expressionsof Caspase-8and DR5in DTDLLM+US group were significantlyup-regulated (P<0.01), and those of Bcl-2were conspicuouslydown-regulated (P <0.01) when compared to other groups.Conclusions DTDLLM+US can significantly inhibit the growth ofliver cancer in nude mice, and the stronger antitumor effect than those innon-targeted DLLM and DTX groups resulted from the stronger inhibitionin tumor angiogenesis and invasion, as well as down-regulated expressionsof Bcl-2and up-regulated expressions of Caspase-8and DR5. DTDLLMcombined with UTMD demonstrates a better targeting ability and strongerantitumor effect than non-targeted docetaxel-loaded microbubbles and pureDTX, and therefore provide a novel, efficient and targeting strategy forliver cancer therapy and ultrasound molecular imaging.更多还原