【作者】 李超；

【导师】 崔大祥；

【作者基本信息】 上海交通大学 ， 生物医学工程（生物纳米医药）， 2015， 博士

【摘要】 胃癌是世界上常见的癌症之一,是继肝癌之后世界上致死率第二的恶性疾病。在中国,平均每年因胃癌死亡的人数约22.9万人,而我国胃癌的早期诊断率不到10%。现代医学要求针对胃癌建立高灵敏度和高特异性的分析与诊断方法,在所有传统及现代的分析方法中,光学成像具有独特的高分辨率优势。过去用于标记组织和细胞的有机荧光染料普遍存在量子产率低、发射光谱宽、易被光漂白等缺点。与传统的有机荧光染料相比,量子点(Quantum dots,QDs)具有许多优异的光谱性能,在生物化学、细胞生物学、分子生物学、分析化学等研究领域显示出极其广阔的应用前景。近年来,随着量子点修饰方法的发展及特异性靶点的发现,量子点已被广泛应用于细胞标记、单分子示踪、活体成像以及疾病靶向诊断和可视化治疗等研究领域。本文以功能化量子点为研究对象,研究了其光谱可控性合成、表面修饰、生物相容性、稳定性及其在胃癌的早期诊断和治疗中的应用,主要包括:胃癌细胞的高灵敏度、高特异性标记,体内肿瘤成像,胃癌疫苗在体内主动靶向免疫治疗及示踪等,具体如下:(1)在非极性溶剂中合成具有核壳结构的CdSe/ZnS量子点,设计制备了两亲性聚合物,该两亲性聚合物具有齿状的烷基配位键,配位键的另一端是具有生物功能的羧基,羧基同时为量子点提供了良好的水溶性基团。使用该聚合物进行CdSe/ZnS量子点的表面化学修饰,成功将量子点由非极性溶剂相转移至水相。评价了经相转移以后的量子点的表面电荷、羧基含量、水合粒径、离子和pH稳定性、体外细胞毒性等参数,为该材料在分子标记、细胞成像及体内示踪应用等,奠定了基础。(2)使用前述相转移后的CdSe/ZnS核壳结构量子点为标准材料,选择BRC AA1和Her2的单克隆抗体作为原型抗体,与量子点进行化学偶联后,制备了抗BRCAA1和抗Her2的两种量子点荧光探针。实验证实,这些探针可以对胃癌细胞MGC-803进行原位特异性靶向标记。此外,使用发射峰657nm量子点标记的MGC-803细胞可以在裸鼠体内通过活体荧光成像系统有效地进行跟踪。结果表明:本研究制备的量子点探针可实现胃癌细胞的体外特异性靶向分子标记,在胃癌模型动物体内可进行高灵敏度、高分辨率成像,在早期胃癌的诊断中具有一定的应用潜力。(3)从健康人类供体的外周血中分离出单个核细胞,在体外经细胞因子诱导,培养出异源树突状细胞。以这种异源性树突状细胞和胃癌细胞为基础,在体外制备了胃癌融合瘤疫苗。本研究合成了具有近红外发射光谱的CuInS2/ZnS核壳结构量子点,按前述方法使用该量子点对融合瘤疫苗进行了标记。将标记后的融合瘤疫苗免疫接种C57BL/6小鼠,用活体成像系统对融合瘤疫苗细胞进行了实时示踪,以评价融合瘤疫苗在体内的免疫治疗和免疫预防效果。结果显示,融合瘤疫苗细胞在体外对多种胃癌细胞系均具有较强的CTL杀伤效应,且可以主动靶向至肿瘤部位,从而实现活体肿瘤成像;另外,当使用适当的肿瘤抗原载体及相应的效应细胞(FC+CIK)免疫接种后,可以在实验动物体内引发至少三个月时间的免疫记忆功能。本研究结果表明:以树突状细胞为基础的胃癌融合瘤疫苗在胃癌的发生、发展、转移及预后等方面,具有良好的应用潜能。(4)制备了荧光磁性纳米粒子(FMNPs),采用FMNPs标记的诱导多能干细胞(iPS),选择胃癌作为体内靶向标志,系统地研究了FMNPs标记的iPS细胞在体内外与胃癌的相互作用,经体内荧光、磁共振成像及体外交变磁场作用下的产热效应等方法,确认了FMNPs标记的iPS细胞可以作为一种有良好治疗作用的生物制剂,可应用于胃癌的体内成像和靶向性热疗,有望推广至其他肿瘤的临床研究或治疗。更多还原

【Abstract】 Gastric cancer(GC) is the fourth most common cancer and the second leading cause of cancer-related death worldwide. In China, the average death number due to GC is about 229 thousand each year, and early diagnosis of GC is less than 10%. It has been proved that, if the strategies of early diagnosis and intervention can be applied to GC, the poor prognosis and 5-year survival rates can be greatly improved.In order to meet the increasing requirements of life and health, modern medicine requires the establishment of new diagnostic methods with high sensitivity and high specificity for GC. However, the traditional organic dyes often failed to label tissues and cells for its low quantum yield, broad emission spectrum, photobleaching and other shortcomings. Compared with traditional organic reagents, quantum dots(QDs) have many inherent properties and have shown the promising potentials in many biological fields, including biochemistry, cell biology, molecular biology, analytical chemistry and other areas of research. In the last decade, with the developments of QDs surface chemistry and the discovery of bio-specific targets, QDs have been widely used in cell labeling, single molecule tracking, and cell tracing, visualized diagnosis and targeted therapy in vivo.In this paper, we chose the functionalized QDs as the research object, intensively investigated the spectral controllable synthesis, surface chemistry, biocompatibility, and stability of QDs. Furthermore, the primary applications of QDs in highly sensitive and specific labeling of GC cells in vitro, in vivo GC imaging with high resolution, the dentritic cell based GC vaccine in active targeted immunotherapy and immunoprophylaxis were investigated systematically, and a series of results were achieved, as follows:1. Firstly, we synthesized the CdSe/ZnS core-shell QDs in organic solvents at high temperature, designed and prepared amphiphilic polymer with alkyl dentate coordination bonds and carboxyl groups. Then, the amphiphilic polymer was used to transfer QDs from orgainc phase to inorganic phase by surface group interaction. Finally, we evaluated the characteristics of the phase transferred QDs, including the surface charge, carboxyl content, hydrated diameter, ionic and pH stability, cytotoxicity in vitro, etc. These data are very critical parameters of QDs in biomolecule labeling, cell imaging and tracing in vivo.2. We selected the aforementioned water-soluble CdSe/ZnS QDs as the standard material, and synthesized the BRCAA1-QDs and Her2-QDs fluorescent probes by chemical conjugation. The results confirmed that these probes can be used to label the GC cell of MGC-803 in vitro sensitively and specifically. In addition, the MGC-803 cells labeled with the 657 nm QDs can be effectively traced in GC model mice by in vivo imaging system. These results indicated that the QDs probes prepared in this study can be introduced to label the GC cells in single molecule level, and can be imaged with high resolution and sensitivity in vivo, showing great potential in diagnosis of early GC.3. We isolated the mononuclear cells from peripheral blood lymphocytes(PBL) of healthy human donors and induced the allogeneic dentritic cells(DCs) by cytokines in vitro. Based on the allogeneic DCs and MGC-803 cells, we prepared the fused GC vaccine cells(FCs) by polyethylene glycol(PEG) mediated fusion. Meanwhile, we synthesized CuInS2/ZnS QDs with emission wavelength in near infrared region(NIR), and functionalized the NIR-QDs with DSPE-PEG. In order to evaluate the effectiveness of immunotherapy and immunoprophylaxis of FCs, we vaccinated the C57BL/6 mouse with NIR-QDs probes labeled FCs and monitored the real-time distribution of the labeled cell in vivo. The results showed that the prepared FCs strongly manifested tumor antigen-specific CTL responses to a wide range of GC cell lines. Additionally, the immune memory research revealed that immunized with appropriate antigen carrier and effector cells(FC+CIKs) in tumor bearing mice can triggered and maintained the corresponding immune memory for a long period(at least 3 months). Efficacy comparison tests in tumor-free and tumor-bear mice demonstrated that the FCs vaccination have the appreciable effects on the occurrence, development, metastasis and prognosis of GC. Our results provide a new insight into the immunobiology of tumor-associated vaccines and offer the proof-of-principle of the application of DCs based vaccine in GC immunotherapy.4. In this part, we labeled induced pluripotent stem cells(iPSCs) with synthesized fluorescent magnetic nanopaticles(FMNPs) and selected the GC as the target in vivo. The interaction of FMNPs-labeled iPSCs and GC were investigated by various methods, including in vivo fluorescence and magnetic resonance imaging(MRI), and the hyperthermal effect coupled with the external magnetic field. The results confirmed that the FMNPs-labeled iPSCs can be an ideal biological agent in GC imaging and targeted hyperthermal treatment. These results are expected to be extended to clinical diagnostic and therapeutic applications of other tumors.更多还原