本文選題：多藥耐藥　切入點(diǎn)：生物素親和素系統(tǒng)　出處：《江南大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：已上市藥物的藥理學(xué)解釋的關(guān)鍵涉及藥物作用靶點(diǎn)的確認(rèn),研究方法主要是設(shè)計(jì)和獲得保留藥物作用的探針分子,進(jìn)行生物材料中分析相應(yīng)藥物靶點(diǎn)。本研究以臨床治療腫瘤一線藥物阿霉素(ADM)和紫杉醇(PTX)為研究材料,設(shè)計(jì)生物素化藥物分子,通過親和素組合形成藥物探針分子,從乳腺導(dǎo)管上皮癌MCF-7細(xì)胞系的耐ADM細(xì)胞(MCF-7/ADM)、PTX細(xì)胞(MCF-7/PTX)中獲得相應(yīng)的藥物結(jié)合蛋白,探討MCF-7細(xì)胞的耐藥機(jī)制。主要結(jié)果如下:1、計(jì)算機(jī)輔助設(shè)計(jì)保持藥物識(shí)別功能的生物素-C6-藥物的連接結(jié)構(gòu),在篩選中找到簡(jiǎn)便的一步法合成中間體生物素-氨基己酸(產(chǎn)物結(jié)構(gòu)經(jīng)核磁圖譜確證,純度為94.65%),并采取DCC,DMAP催化法合成獲得生物素化ADM與生物素化PTX兩種藥物類似物,經(jīng)1H NMR和HPLC確認(rèn)結(jié)構(gòu)及純度后,測(cè)試藥物類似物對(duì)細(xì)胞生長(zhǎng)抑制情況及其在細(xì)胞中的分布并與原藥對(duì)比:生物素化ADM和生物素化PTX干預(yù)MCF-7、MCF-7/ADM以及MCF-7/PTX的IC50沒有明顯的改變:當(dāng)作用于MCF-7/W細(xì)胞,生物素化ADM的IC50值為8.554μM,ADM的IC50值為8.656μM,對(duì)于MCF-7/ADM細(xì)胞,生物素化ADM的IC50值為127.2μM,ADM的IC50值為104.5μM;當(dāng)作用于MCF-7/W細(xì)胞,生物素化PTX的IC50值為10.17μM,PTX的IC50值為12.01μM;對(duì)于MCF-7/PTX細(xì)胞,生物素化PTX的IC50值為82.16μM,PTX的IC50值為93.26μM;激光共聚焦成像亦驗(yàn)證了其在細(xì)胞內(nèi)的分布和作用方式?jīng)]有明確的變化,說明生物素碳鏈結(jié)構(gòu)的引入不會(huì)干擾ADM或PTX的藥物作用。2、以生物素化ADM和生物素化PTX藥物類似物為探針,與包被鏈霉親和素的磁珠組建可快速分離藥物探針分子,以MCF-7/ADM與MCF-7/PTX細(xì)胞裂解液為研究材料,分別采用生物素化ADM和生物素化PTX藥物類似物-親和素磁珠分析相應(yīng)的結(jié)合蛋白。經(jīng)電泳分離,Q TOF-MS檢測(cè)蛋白序列并與數(shù)據(jù)庫(kù)對(duì)比,獲得對(duì)ADM耐藥性狀相關(guān)的蛋白Junction plakoglobin和Hyaluronidase,以及與PTX耐藥性狀相關(guān)的蛋白Glutathione S-transferase P,Peroxiredoxin-1等二十余種蛋白。本研究構(gòu)建了具有一定通量的分析藥物結(jié)合蛋白的方法,為篩選和確認(rèn)藥物作用靶點(diǎn)提供了新的可行的工具,有助于解析相應(yīng)藥物的作用機(jī)制和耐藥機(jī)理。
[Abstract]:The key to the pharmacological interpretation of marketed drugs involves the identification of drug action targets, primarily by designing and obtaining probe molecules that retain the effects of drugs. Using adriamycin adriamycin (adriamycin) and paclitaxel (PTX) as the research materials, the biotin drug molecules were designed, and the drug probe molecules were formed by the combination of avidin, adriamycin (adriamycin) and paclitaxel (PTX). The corresponding drug-binding protein was obtained from MCF-7 / ADMN-PTX cell line of breast ductal carcinoma MCF-7 cell line, which was resistant to ADM, and the corresponding drug binding protein was obtained from MCF-7 / PTX cell line. To study the mechanism of drug resistance in MCF-7 cells. The main results are as follows: 1. Computer aided design of the connective structure of biotin -C6- drugs, which maintains drug recognition function, A simple one-step method was found for the synthesis of biotinylhexanoic acid (the structure of the product was confirmed by NMR spectra, the purity was 94.65g), and the biotinylated ADM and biotinylated PTX analogues were synthesized by DCC-DMAP catalytic method. The structure and purity were confirmed by 1H NMR and HPLC. The inhibition of drug analogues on cell growth and their distribution in cells were measured and compared with the original drug. Biotin ADM and biotinylated PTX did not change significantly in MCF-7 / 7 / ADM and MCF-7/PTX IC50: they were used in MCF-7/W cells. The IC50 value of biotinylated ADM was 8.554 渭 M, the IC50 value of biotin ADM was 8.656 渭 M, the IC50 value of biotin ADM was 127.2 渭 M, the IC50 value of biotinylated PTX was 104.5 渭 M, the IC50 value of biotinylated PTX was 12.01 渭 M for MCF-7/W cells, and the IC50 value for MCF-7/PTX cells was 10.17 渭 m. The IC50 value of biotinylated PTX was 82.16 渭 MPTX and the IC50 value of biotinylated PTX was 93.26 渭 M. Laser confocal imaging also proved that there was no clear change in the distribution and mode of action in the cells. It was suggested that the introduction of biotinylated carbon chain structure would not interfere with the drug action of ADM or PTX. Using biotinylated ADM and biotinylated PTX analogues as probes and magnetic beads coated with streptavidin, the drug probe molecules could be rapidly separated. Using MCF-7/ADM and MCF-7/PTX cell lysate as materials, the binding proteins were analyzed by biotin ADM and avidin magnetic beads respectively. The protein sequences were detected by electrophoresis and compared with the database. More than 20 proteins, such as Junction plakoglobin and Hyaluronidase associated with drug resistance to ADM, and Glutathione S-transferase P#en0# peroxiredoxin-1 were obtained. It provides a new and feasible tool for screening and confirming drug action targets, and helps to analyze the mechanism of drug resistance and action.
【學(xué)位授予單位】：江南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R96

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