共载阿霉素和pTRAIL的硫辛酸多肽胶束构建及其抗耐药乳腺癌研究
发布时间：2018-11-03 16:46
【摘要】：乳腺癌的发病率不断上升,目前已成为严重危害女性健康的重要肿瘤之一,临床治疗中常因乳腺癌细胞产生多药耐药性而导致化疗的失败[2]。因此,研究如何增强耐药乳腺癌细胞的化疗敏感性是一个极具挑战性的课题。本课题通过广谱抗肿瘤药阿霉素与基因药物pTRAIL联用,以硫辛酸多肽作为载体,研究其抗耐药乳腺癌的体内外作用。本课题的第一部分报道了共载阿霉素和pTRAIL的硫辛酸多肽纳米胶束构建及其表征。通过多肽固相合成法合成了含有聚精氨酸、组氨酸和硫辛酸的多肽单体,通过制备液相进行纯化以及高效液相色谱和质谱进行分析,显示制备的多肽单体符合实验要求。利用硫辛酸分子内二硫键在半胱氨酸催化下可进行分子间交联,形成多肽聚合物。核磁共振氢谱及凝胶渗透色谱鉴定聚合物合成成功。将多肽聚合物与脂溶性阿霉素在超声乳化条件下自组装形成多肽胶束,并使其外部富含阳离子部分与基因药物pTRAIL通过静电相互作用结合,制备成共载阿霉素和pTRAIL的多肽纳米胶束。对多肽胶束进行表征,结果显示,粒径为69±2.14nm,电位为30.7±2.94 mV,载药量为12.5%。本课题第二部分报道了共载阿霉素和pTRAIL的硫辛酸多肽胶束抗耐药乳腺癌的体外评价。与单纯阿霉素相比,硫辛酸修饰的多肽胶束可以被耐药乳腺癌细胞株MCF-7/ADR快速摄取,阿霉素释放进入细胞核。同时,pTRAIL在细胞质中富集。细胞毒性实验表明,硫辛酸多肽可以有效介导阿霉素和pTRAIL进入细胞,抑制耐药乳腺癌细胞的增值,促进细胞凋亡。本课题的第三部分报道了共载阿霉素和pTRAIL的多肽纳米胶束抗耐药乳腺癌的体内作用。成功构建了MCF-7/ADR耐药乳腺癌裸鼠皮下移植瘤模型,通过小动物成像系统观察荧光标记的多肽胶束在裸鼠体内的分布情况,结果显示多肽胶束能较好地富集于肿瘤部位,实现靶向性。肿瘤生长抑制实验和裸鼠体重变化情况及肿瘤组织HE染色结果显示,共载阿霉素和pTRAIL的多肽胶束可以抑制肿瘤的生长,且阿霉素广泛的心脏毒性在多肽胶束组未见,表明其具有较好的安全性。本课题构建了共载化疗药阿霉素与基因药pTRAIL的多肽胶束载体,为临床治疗耐药乳腺癌和其他肿瘤提供了参考与借鉴。
[Abstract]:The incidence of breast cancer is on the rise, and it has become one of the most important tumors that seriously endanger the health of women. The failure of chemotherapy is often caused by the multidrug resistance of breast cancer cells in clinical treatment [2]. Therefore, it is a challenging task to study how to enhance chemosensitivity of drug-resistant breast cancer cells. The aim of this study was to study the in vitro and in vivo effects of antitumor drug doxorubicin and gene drug pTRAIL using lipoic acid polypeptide as a carrier. In the first part of this paper, we report the construction and characterization of lipoic acid polypeptide nanomicelles containing adriamycin and pTRAIL. Polypeptide monomers containing polyarginine, histidine and lipoic acid were synthesized by solid phase polypeptide synthesis. The polypeptide monomers were purified by preparing liquid phase and analyzed by high performance liquid chromatography and mass spectrometry. The intramolecular disulfide bond of lipoic acid can be cross-linked with cysteine to form polypeptide polymer. The synthesis of polymer was confirmed by NMR and gel permeation chromatography. The polypeptide polymer and lipophilic adriamycin were self-assembled to form polypeptide micelles under phacoemulsification, and the external cationic fraction was bound to the gene drug pTRAIL by electrostatic interaction. Polypeptide nanomicelles co-loaded with adriamycin and pTRAIL were prepared. The results showed that the particle size of polypeptide micelles was 69 卤2.14 nm and the potential was 30.7 卤2.94 mV,. In the second part of this study, the in vitro evaluation of drug-resistant breast cancer by lipoic acid polypeptide loaded with adriamycin and pTRAIL was reported. Compared with adriamycin alone, lipoxylic acid modified polypeptide micelles could be rapidly ingested by drug-resistant breast cancer cell line MCF-7/ADR, and adriamycin released into the nucleus. Meanwhile, pTRAIL was enriched in cytoplasm. Cytotoxicity test showed that lipoic acid polypeptide could effectively mediate adriamycin and pTRAIL into the cells, inhibit the proliferation of drug-resistant breast cancer cells, and promote cell apoptosis. In the third part of this study, we reported the anti-drug-resistant breast cancer in vivo by polypeptide nanomicelles loaded with adriamycin and pTRAIL. The subcutaneous transplanted tumor model of MCF-7/ADR resistant breast cancer in nude mice was successfully constructed. The distribution of fluorescent labeled polypeptide micelles in nude mice was observed by small animal imaging system. The results showed that polypeptide micelles could be well enriched in tumor sites. Achieve targeting. Tumor growth inhibition test, weight change of nude mice and HE staining of tumor tissue showed that the polypeptide micelles carrying adriamycin and pTRAIL could inhibit the growth of tumor, and the extensive cardiac toxicity of adriamycin was not found in polypeptide micelle group. It shows that it has better safety. A polypeptide micelle vector carrying adriamycin and pTRAIL was constructed to provide reference for clinical treatment of drug-resistant breast cancer and other tumors.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R943;R96
，
本文编号：2308358
[Abstract]:The incidence of breast cancer is on the rise, and it has become one of the most important tumors that seriously endanger the health of women. The failure of chemotherapy is often caused by the multidrug resistance of breast cancer cells in clinical treatment [2]. Therefore, it is a challenging task to study how to enhance chemosensitivity of drug-resistant breast cancer cells. The aim of this study was to study the in vitro and in vivo effects of antitumor drug doxorubicin and gene drug pTRAIL using lipoic acid polypeptide as a carrier. In the first part of this paper, we report the construction and characterization of lipoic acid polypeptide nanomicelles containing adriamycin and pTRAIL. Polypeptide monomers containing polyarginine, histidine and lipoic acid were synthesized by solid phase polypeptide synthesis. The polypeptide monomers were purified by preparing liquid phase and analyzed by high performance liquid chromatography and mass spectrometry. The intramolecular disulfide bond of lipoic acid can be cross-linked with cysteine to form polypeptide polymer. The synthesis of polymer was confirmed by NMR and gel permeation chromatography. The polypeptide polymer and lipophilic adriamycin were self-assembled to form polypeptide micelles under phacoemulsification, and the external cationic fraction was bound to the gene drug pTRAIL by electrostatic interaction. Polypeptide nanomicelles co-loaded with adriamycin and pTRAIL were prepared. The results showed that the particle size of polypeptide micelles was 69 卤2.14 nm and the potential was 30.7 卤2.94 mV,. In the second part of this study, the in vitro evaluation of drug-resistant breast cancer by lipoic acid polypeptide loaded with adriamycin and pTRAIL was reported. Compared with adriamycin alone, lipoxylic acid modified polypeptide micelles could be rapidly ingested by drug-resistant breast cancer cell line MCF-7/ADR, and adriamycin released into the nucleus. Meanwhile, pTRAIL was enriched in cytoplasm. Cytotoxicity test showed that lipoic acid polypeptide could effectively mediate adriamycin and pTRAIL into the cells, inhibit the proliferation of drug-resistant breast cancer cells, and promote cell apoptosis. In the third part of this study, we reported the anti-drug-resistant breast cancer in vivo by polypeptide nanomicelles loaded with adriamycin and pTRAIL. The subcutaneous transplanted tumor model of MCF-7/ADR resistant breast cancer in nude mice was successfully constructed. The distribution of fluorescent labeled polypeptide micelles in nude mice was observed by small animal imaging system. The results showed that polypeptide micelles could be well enriched in tumor sites. Achieve targeting. Tumor growth inhibition test, weight change of nude mice and HE staining of tumor tissue showed that the polypeptide micelles carrying adriamycin and pTRAIL could inhibit the growth of tumor, and the extensive cardiac toxicity of adriamycin was not found in polypeptide micelle group. It shows that it has better safety. A polypeptide micelle vector carrying adriamycin and pTRAIL was constructed to provide reference for clinical treatment of drug-resistant breast cancer and other tumors.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R943;R96
，
本文编号：2308358
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