發(fā)布時間：2018-11-03 16:46

【摘要】：乳腺癌的發(fā)病率不斷上升,目前已成為嚴重危害女性健康的重要腫瘤之一,臨床治療中常因乳腺癌細胞產生多藥耐藥性而導致化療的失敗[2]。因此,研究如何增強耐藥乳腺癌細胞的化療敏感性是一個極具挑戰(zhàn)性的課題。本課題通過廣譜抗腫瘤藥阿霉素與基因藥物pTRAIL聯用,以硫辛酸多肽作為載體,研究其抗耐藥乳腺癌的體內外作用。本課題的第一部分報道了共載阿霉素和pTRAIL的硫辛酸多肽納米膠束構建及其表征。通過多肽固相合成法合成了含有聚精氨酸、組氨酸和硫辛酸的多肽單體,通過制備液相進行純化以及高效液相色譜和質譜進行分析,顯示制備的多肽單體符合實驗要求。利用硫辛酸分子內二硫鍵在半胱氨酸催化下可進行分子間交聯,形成多肽聚合物。核磁共振氫譜及凝膠滲透色譜鑒定聚合物合成成功。將多肽聚合物與脂溶性阿霉素在超聲乳化條件下自組裝形成多肽膠束,并使其外部富含陽離子部分與基因藥物pTRAIL通過靜電相互作用結合,制備成共載阿霉素和pTRAIL的多肽納米膠束。對多肽膠束進行表征,結果顯示,粒徑為69±2.14nm,電位為30.7±2.94 mV,載藥量為12.5%。本課題第二部分報道了共載阿霉素和pTRAIL的硫辛酸多肽膠束抗耐藥乳腺癌的體外評價。與單純阿霉素相比,硫辛酸修飾的多肽膠束可以被耐藥乳腺癌細胞株MCF-7/ADR快速攝取,阿霉素釋放進入細胞核。同時,pTRAIL在細胞質中富集。細胞毒性實驗表明,硫辛酸多肽可以有效介導阿霉素和pTRAIL進入細胞,抑制耐藥乳腺癌細胞的增值,促進細胞凋亡。本課題的第三部分報道了共載阿霉素和pTRAIL的多肽納米膠束抗耐藥乳腺癌的體內作用。成功構建了MCF-7/ADR耐藥乳腺癌裸鼠皮下移植瘤模型,通過小動物成像系統(tǒng)觀察熒光標記的多肽膠束在裸鼠體內的分布情況,結果顯示多肽膠束能較好地富集于腫瘤部位,實現靶向性。腫瘤生長抑制實驗和裸鼠體重變化情況及腫瘤組織HE染色結果顯示,共載阿霉素和pTRAIL的多肽膠束可以抑制腫瘤的生長,且阿霉素廣泛的心臟毒性在多肽膠束組未見,表明其具有較好的安全性。本課題構建了共載化療藥阿霉素與基因藥pTRAIL的多肽膠束載體,為臨床治療耐藥乳腺癌和其他腫瘤提供了參考與借鑒。
[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 鹵2.14 nm and the potential was 30.7 鹵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.
【學位授予單位】：第二軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R943;R96
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本文編號：2308358