【作者】 贾艳辉；

【导师】 陈继营； 袁玫； 郭全义；

【作者基本信息】 中国人民解放军军医进修学院 ， 外科学， 2010， 硕士

【摘要】 研究背景：阿霉素(Adriamycin, ADR或Doxorubicin, DOX)是一种蒽环类广谱抗肿瘤药,具有强烈的细胞毒性,可广泛应用于治疗骨肉瘤、白血病、淋巴瘤、卵巢癌及晚期乳腺癌。但在化疗过程中,阿霉素对肿瘤组织特异性较差,可引起剂量限制性毒性。而且肿瘤细胞也可对多种化疗药物产生抗药性。这些问题一直限制着其在肿瘤治疗中的应用。为了解决这些问题,近几十年来的研究主要集中于研发肿瘤特异性药物及载体系统,可将抗肿瘤药物选择性地集中释放到肿瘤部位。纳米技术的进步为肿瘤靶向给药系统的进一步发展提供了广阔的发展前景。纳米靶向给药一般采用两种形式,即被动靶向及主动靶向。被动靶向通过肿瘤组织血管的高通透性和不连续性导致纳米颗粒渗漏到血管外肿瘤组织及肿瘤限制淋巴引流系统这两种途径,实现纳米颗粒在肿瘤组织的选择性聚集,称为增强的透过及滞留(EPR)效应。主动靶向主要利用肿瘤细胞表面过度表达的特异性抗原表位和受体作为靶点,或通过纳米颗粒的某种物理性质(如对温度、酸碱度、电荷、光、声或磁场的灵敏度)实现其靶向作用。纳米颗粒作为靶向给药系统具有下列显著优势：(1)能够实现多重靶向机制,增强药物对肿瘤组织的特异性及选择性；(2)降低达到靶区特定浓度所需的药物剂量；(3)降低正常组织的药物浓度,减轻毒副作用；(4)通过胞吞作用或吞噬作用可在细胞水平发挥药效；(5)可以构建多功能纳米颗粒,集肿瘤显像、靶向化疗、热疗、放疗于一体。研究目的：本课题的目的是构建一种多重靶向的纳米给药系统一偶联单克隆抗体的载阿霉素磁性纳米颗粒,在外部磁场、抗体靶向及纳米颗粒被动靶向共同作用下,能选择性地将阿霉素释放到肿瘤组织,降低用药剂量及毒副作用。本阶段的研究内容主要包括两方面：(1)体外研究两种不同粒径的载阿霉素的磁性纳米颗粒的细胞毒性；(2)制备、纯化并鉴定抗人骨肉瘤OS-732细胞株的单克隆抗体。研究方法：(1)本实验中采用两种不同粒径的载阿霉素的磁性纳米颗粒：Fe3O4-DEX-DOX及Fe3O4-PLGA-DOX,以比较它们对细胞的毒性；Hoechst33258染色,荧光显微镜观察其被细胞摄取及在细胞内分布的情况；MTT法检测其对肿瘤细胞的增殖抑制作用；LDH释放法测定其对肿瘤细胞的杀伤作用：Annexin V-FITC/PI双荧光染色,流式细胞术(FCM)检测其促肿瘤细胞凋亡／坏死的作用。(2)利用杂交瘤细胞诱生腹水制备单克隆抗体,Protein A Sepharose CL-4B亲和层析法纯化单克隆抗体,聚丙烯酰胺凝胶电泳(SDS-PAGE)、免疫组化染色及酶联免疫特异性测定(ELISA)法鉴定其性质。研究结果：(1)荧光显微镜观察发现,载阿霉素磁性纳米颗粒可选择性地大量进入肿瘤细胞,并可进入细胞核,却很少进入正常人胚肺细胞。(2)载阿霉素磁性纳米颗粒具有逆转肿瘤细胞多药耐药性的作用。(3)Annexin V-FITC/PI双荧光染色,流式细胞术检测结果显示,载阿霉素的磁性纳米颗粒促进肿瘤细胞坏死,坏死率高于单纯阿霉素,随浓度增高及时间延长,凋亡率下降,坏死率增高。(4)MTT检测结果显示,载阿霉素磁性纳米颗粒抑制肿瘤细胞生长的作用强于单纯阿霉素,差异有统计学意义(p<0.05)。(5)LDH检测结果显示,载阿霉素的磁性纳米颗粒组释放LDH显著高于单纯阿霉素组(p<0.05)。(6)载阿霉素磁性纳米颗粒易被单核巨噬细胞及人脐静脉内皮细胞摄取,提示给药途径应尽量避免与血液循环系统接触。(7)纯化的单克隆抗体对人骨肉瘤、软骨肉瘤、脂肪肉瘤、尤文氏肉瘤及恶性纤维组织细胞瘤等骨科恶性肿瘤的组织切片免疫组化染色阳性,SDS-PAGE鉴定抗体纯度高达93%,ELISA测定活性为1X10-7。结论：(1)载阿霉素的磁性纳米颗粒能够选择性地进入并存留在肿瘤细胞内,能增强阿霉素对肿瘤细胞的毒性,促进肿瘤细胞坏死。(2)制备、纯化的单克隆抗体特异性强,能与多种骨科恶性肿瘤细胞结合,纯度高,生物活性好。更多还原

【Abstract】 Background:The anthracycline antibiotic adriamycin or doxorubicin is a highly efficient antineoplastic agent commonly used in the therapy of a variety of cancers like osteosarcoma, leukaemia, lymphomas, ovarian cancer and late stage breast cancer. During chemotherapy, however, pharmacologically active doxorubicin reaches the tumor tissue with poor specificity and can induce dose-limiting toxicity. Moreover, the cancer cells may eventually develop resistance to multiple chemotherapeutics. These problems have long been primary hindrances for the clinical application of doxorubicin. To tackle these difficulties, decades of research have focused on developing cancer-specific drugs or delivery systems that can selectively localise chemotherapeutics to the tumor site. Recent advances in nanotechnology promise further developments in tumor targeted drug delivery systems. Nanoparticle-based targeted drug delivery may use passive or active strategies. Passive targeting occurs as a result of extravasation of the nanoparticles at the tumor site where the microvasculature is hyperpermeable and leaky, a process aided by tumor-limited lymphatic drainage. Combined, these factors lead to the selective accumulation of nanoparticles in tumor tissue, a phenomenon known as enhanced permeability and retention (EPR) effect. Active targeting is based on the over or exclusive expression of different epitopes or receptors in tumor cells, and on specific physical characteristics (e.g. sensitivity to temperature, pH, electric charge, light, sound or magnetism). The potential of nanoparticle-based drug delivery systems stems from significant advantages such as:(1) the ability to achieve multiple target access allowing for even better specificity and selectivity to the tumor mass; (2) the reduction of the quantity of drug needed to attain a particular concentration in the vicinity of the target; (3) the reduction of the drug concentration at normal tissue, minimizing severe side effects; (4) the ability to act at the cellular level through endocytosis or phagocytosis; (5) the capability to creat multifunctional nanoparticle formulations combining tumor imaging, drug targeting, guided hyperthermia and radiation in an all-in-one system.Objective:The purpose of the present study is to construct a multiple targeted drug delivery system-doxorubicin loaded magnetic Fe3O4 nanoparticles with monoclonal antibody conjugated to the surface, which can selectively target nanoparticles to the tumor mass under the co-ordination of magnetic field, antibody and nanoparticle-based targeting mechanisms, thus reducing overall dosage and side effects. At the present stage, the study consists of two parts:(1) in vitro study of the cytotoxicity of two kinds of doxorubicin loaded magnetic nanoparticles with different particle sizes; (2) preparation, purification and identification of the monoclonal antibody against human osteosarcoma OS-732 cell line.Methods:(1)Two kinds of doxorubicin loaded magnetic Fe3O4 nanoparticles (Fe3O4-DEX-DOX and Fe3O4-PLGA-DOX) with different sizes were prepared and utilized in the present study to compare their cytotoxic effects on cancer cells. The celluar uptake and distribution of doxorubicin loaded magnetic nanoparticles were observed by fluorescence microscopy; The inhibitory effects on the proliferation of cancer cells were evaluated in vitro by MTT assay; The cytotoxic effects on the membrane damage of cancer cells were evaluated in vitro by lactate dehydrogenase (LDH) assay; The apoptotic and necrotic rates of cancer cells exposed to doxorubicin loaded magnetic nanoparticles were determined by flow cytometry using the Annexin V-FITC/PI staining method. (2)The monoclonal antibody against human osteosarcoma OS-732 cell line was prepared through ascites induced by hybridoma cells, and purified by Protein A Sepharose CL-4B affinity chromatography and its properties were evaluated by SDS-PAGE、Immunohistochemistry and ELISA.Results:(1)The observation of fluorescence microscopy demonstrated that doxorubicin loaded magnetic nanoparticles faciliated internalization of doxorubicin to cancer cells with selectivity, and particularly may penentrate the nuclear membrane, however with much less uptake by human embryonic lung cells. (2)Doxorubicin loaded magnetic nanoparticles showed great potential to reverse multidrug resistance of cancer cells. (3)The FCM results illustrated that magnetic nanoparticles loaded with doxorubicin induced higher necrotic rates than free doxorubicin, and the necrotic rates increased in a dose and time dependent manner. (4) The MTT results indicated that the inhibitory effects of magnetic nanoparticles loaded with doxorubicin on cell proliferation were higher than that of free doxorubicin, with statistical significance (p<0.05). (5)The LDH leakages in groups exposed to magnetic nanoparticles loaded with doxorubicin were significantly higher than that of free doxorubicin group(p<0.05). (6) Magnetic nanoparticles loaded with doxorubicin were preferrentially ready for uptake by the mononuclear macrophages and human umbilical vascular endothelial cells, suggesting that the administration routes should be separated from circulation system. (7)With immunohistochemical staining, the monoclonal antibody showed positive reactions on formaldehyde-fixed sections from human osteosarcoma, chondrosarcoma, liposarcoma, Ewing’s sarcoma, malignant fibrous histiotoma and so on; the purity of monoclonal antibody was identified about 93%with SDS-PAGE (10%); the immunoactivity was 1 X 10-7 by ELISA.Conclusions:(1)Magnetic nanoparticles loaded with doxorubicin can faciliate penetration and retention in cancer cells selectively, enhance cytotoxicity of doxorubicin, and induce necrosis instead of apoptosis. (2)The monoclonal antibodies produced are of high purity and immunoactivity with specificity to malignant bone tumors.更多还原