【作者】 费丹；

【导师】 高忠礼；

【作者基本信息】 吉林大学 ， 骨外科， 2014， 博士

【摘要】 表皮生长因子受体（epidermal growth factor receptor, EGFR）对于多种细胞具有促进增殖和营养作用。EGFR在多种肿瘤细胞内高表达，说明其与这些肿瘤的发生、发展等变化有关。另外，大量研究表明EGFR信号系统与肿瘤的发生和肿瘤耐药性关系密切，并且EGFR的异常变化也是骨肉瘤的显著特点。蜂毒（Beevenom, BV）做为免疫相关疾病治疗制剂已广泛应用，特别是在肿瘤治疗领域其作用十分显著。研究发现，肾癌、肺癌、肝癌、前列腺癌、膀胱癌、乳腺癌以及白血病细胞均可成为BV主要成分蜂毒肽（Melittin）的靶标。Melittin激活PLA2从而发挥细胞毒作用已被公认为BV抗肿瘤的主要作用机制。Melittin通过激活Caspase酶和基质金属蛋白酶，诱导多种肿瘤细胞发生凋亡，从而实现其抗肿瘤作用。现已证明，将Melittin与激素受体、特异性抗体等多种具有导向作用的物质融合表达，可以有效治疗多种肿瘤。本研究将Melittin通过柔性肽与抗EGFRscFv连接，并由大肠杆菌进行表达，制备了融合蛋白antiEGFR/MEL。对表达条件进行了优化，并进行了复性和初步纯化。利用表达的融合蛋白，针对人骨肉瘤细胞进行了体内、外抑瘤实验研究。研究结果表明，含有EGFR单链抗体的重组蛋白antiEGFR/MEL可与OS732细胞有效结合，并定位于OS732细胞膜表面。antiEGFR/MEL能够有效抑制OS732细胞的生长，但对正常细胞L02无明显作用。另外，antiEGFR/MEL抑制OS732细胞生长的现象，呈现明显的时间效应和剂量效应趋势。本研究还利用BALB/c小鼠结合小鼠骨肉瘤细胞S180，建立了实体肉瘤细胞模型，并开展了体内抑瘤实验研究。研究结果表明，antiEGFR/MEL可以抑制S180实体肿瘤生长、延长动物模型生存期、延长动物模型肿瘤倍增时间和肿瘤生长延迟时间。以上结果说明，antiEGFR/MEL具有应用于骨肉瘤生物治疗研究的潜质。更多还原

【Abstract】 Osteosarcoma (OS) is the most common sarcomas in children and adolescents.OS is the most frequent bone cancer occurring mostly in children and adolescentsbetween10and20years of age. It is thought to arise from mesenchymal boneforming tissue and its histological hallmark is the production of malignant osteoid.The metaphyseal growth regions of the long bones of the extremities are the mostcommon site of the primary bone tumor with peak incidences following theadolescent growth spurt and the seventh and eighth decades of life. Currentmultimodal therapy consisting of surgery and chemotherapy, achieves a5-yearsurvival rate of approximately60–70%in children while patients who present withmetastatic disease at diagnosis have a10–30%survival rate. The chemotherapy agentswhich have long demonstrated anti-tumor activity in OS include cisplatin,doxorubicin, ifosfamide, and high dose-methotrexate. The use of novel effectivetherapeutic approaches and treatment strategies in patients who are resistant to currenttherapy could provide an improvement in outcome in patients.In more than20years of drug development, agents targeting members of thehuman epidermal growth factor receptor family—EGFR (also known as HER-1,erbB1) and HER-2/neu (also known as erbB2)—have shown encouraging therapeuticefficacy. The first to be approved by the US Food and Drug Administration (FDA) in1998was trastuzumab (Herceptin) for the treatment of HER-2(erbB-2)-positivebreast cancer. Over the past few years, four EGFR specific agents have also receivedregulatory approval. Cetuximab (Erbitux) for metastatic colorectal cancer (mCRC)and squamous cell carcinoma of the head and neck (SCCHN), Gefitinib (Iressa) foradvanced or metastatic non-small-cell lung cancer (mNSCLC), Erlotinib (Tarceva) foradvanced or metastatic pancreatic cancer and non-small-cell lung cancer (NSCLC),and Panitumumab (Vectibix) for metastatic colorectal cancer (mCRC).Bee venom is a unique weapon for the bee colony and composed of melittin,apamin, adolapin, mast cell degranulating peptide, phospholipase A2, hyaluronidase,and some non-peptide components (histamine, dopamine, norepinephrine, etc.).Among them, melittin is the major component of bee venom (50–70%) and is acationic, hemolytic peptide. It is composed of26amino acid residues in which theamino-terminal region (residues1–20) is predominantly hydrophobic whereas the carboxyl-terminal region (residues21–26) is hydrophilic. Its amphiphilic propertymakes it water-soluble and membrane-active lytic peptide ideally suited formonitoring mechanisms of pore formation and lipid–protein interactions inmembranes. Above all, melittin shows several important medical effects includingenhancement of phospholipase A2activity, cytotoxic effects against cancer cells, andanti-inflammatory, anti-microbial, and anti-arthritic effects.In this study, we have fused the anti-EGFR single chain variable fragment (scFv)with melittin aiming at EGFR-targeted therapy for Osteosarcoma. The designed genesequence was optimized, then cloned and expressed in E. coli BL21strains. Inaddition, the expression of the fusion protein was optimized for its in vivo and in vitroantitumoral activity.更多还原