【摘要】：化療是治療癌癥的重要手段之一,但現(xiàn)有抗癌藥物一般具有較大的毒副作用,為了減小其副作用,大量新穎的前藥被開發(fā)出來。診斷治療型前藥,兼?zhèn)淞嗽\斷與治療的功能,實(shí)現(xiàn)了藥物的可控釋放,并對(duì)活性藥物的釋放進(jìn)行檢測,及時(shí)反饋了治療效果。有機(jī)小分子診療前藥具有結(jié)構(gòu)簡單、便于修飾、生物相容性好等特點(diǎn),成為研究的熱點(diǎn)。大多數(shù)腫瘤是實(shí)體瘤,乏氧是實(shí)體瘤的重要特征之一,導(dǎo)致腫瘤對(duì)化療藥物的不敏感并產(chǎn)生耐藥性。然而,乏氧也為腫瘤的治療提供了思路,即用乏氧響應(yīng)基團(tuán)修飾活性藥物,使其只在腫瘤的乏氧區(qū)域被激活,釋放活性藥物,達(dá)到選擇性治療的效果。氟脲苷(FDU)是一種抗代謝類抗腫瘤藥物,用于臨床治療胃癌、乳腺癌等,但其引起的毒副作用(如惡心、嘔吐等)限制了其應(yīng)用。為降低FDU在腫瘤治療過程中產(chǎn)生的毒副作用,并在線檢測藥物在體內(nèi)的分布和積蓄情況,本文以硝基芐基為乏氧響應(yīng)基團(tuán),以2-羧基-2’-羥基聯(lián)苯(BCM)為熒光染料前體,通過共價(jià)鍵與活性藥物FDU進(jìn)行偶聯(lián),設(shè)計(jì)合成了乏氧響應(yīng)型診療前藥FDU-BCM-NO_2。采用NMR、MS和IR等手段對(duì)前藥和中間體進(jìn)行表征;采用HPLC法對(duì)前藥的還原組分進(jìn)行測定,探討其釋藥機(jī)制;采用熒光光譜法測定前藥與Na2S2O4孵育不同時(shí)間后的熒光強(qiáng)度,探討FDU釋放量隨時(shí)間的變化;采用MTT法測試了FDU和FDU-BCM-NO_2對(duì)MCF-7、MGC-803腫瘤細(xì)胞和BRL-3A正常肝細(xì)胞的細(xì)胞毒性,采用流式細(xì)胞分析法測試FDU-BCM-NO_2誘導(dǎo)腫瘤細(xì)胞凋亡的情況,評(píng)價(jià)了FDU-BCM-NO_2的體外抗腫瘤效果;采用熒光成像法對(duì)FDU-BCM-NO_2孵育的乏氧腫瘤細(xì)胞及腫瘤球成像,評(píng)價(jià)了FDU-BCM-NO_2對(duì)乏氧的診斷和對(duì)FDU的示蹤效果。結(jié)果表明:乏氧條件下,FDU-BCM-NO_2在被還原的同時(shí)釋放出活性藥物FDU和熒光分子BCM,BCM釋放量隨乏氧孵育時(shí)間的延長而增加,可利用BCM的熒光信號(hào)監(jiān)測FDU的釋放。在常氧條件下,FDU分別孵育MCF-7乳腺癌細(xì)胞、MGC-803胃癌細(xì)胞和BRL-3A肝細(xì)胞48 h后,細(xì)胞存活率分別為41.43%、41.70%和48.09%,而FDU-BCM-NO_2孵育的三種細(xì)胞存活率則分別為91.09%、91.22%和99.08%;在乏氧條件下,FDU孵育的MCF-7和MGC-803的細(xì)胞存活率則分別為22.57%和41.70%,而采用FDU-BCM-NO_2孵育后,細(xì)胞存活率則分別44.76%和51.27%,表明FDU對(duì)正常細(xì)胞和腫瘤細(xì)胞均有抑制作用,而FDU-BCM-NO_2具有乏氧選擇性,只對(duì)乏氧腫瘤細(xì)胞具有抑制作用,對(duì)正常細(xì)胞則不產(chǎn)生毒性。在乏氧條件下,FDU-BCM-NO_2孵育的MGC-803細(xì)胞,52.21%的細(xì)胞發(fā)生凋亡,表明FDU-BCM-NO_2以凋亡的方式抑制腫瘤細(xì)胞增殖。FDU-BCM-NO_2在乏氧條件下釋放出的BCM具有雙光子激發(fā)特性,其熒光信號(hào)可用于腫瘤細(xì)胞及腫瘤球乏氧程度和藥物釋放的檢測。FDU-BCM-NO_2具有診斷、治療的雙重功能,利用實(shí)體瘤的乏氧特性可實(shí)現(xiàn)FDU的可控釋放,避免了其對(duì)正常細(xì)胞的毒副作用,為實(shí)現(xiàn)腫瘤的個(gè)體化治療提供了新的思路。
[Abstract]:Chemotherapy is one of the important means for the treatment of cancer, but the existing anticancer drugs generally have large toxic and side effects. In order to reduce their side effects, a large number of novel prodrugs have been developed. The diagnostic and therapeutic prodrugs have both the function of diagnosis and treatment, the controlled release of drugs, the detection of the release of active drugs, and timely feedback. The effect of treatment. The drug has the characteristics of simple structure, easy to modify and good biocompatibility, and it has become a hot spot. Most tumors are solid tumors. Hypoxia is one of the important characteristics of solid tumor, which causes the tumor to be insensitive to chemotherapy drugs and produces resistance. However, hypoxia also provides thought for the treatment of tumor. FDU is an antitumor antitumor drug used in the clinical treatment of gastric cancer and breast cancer, but its toxic and side effects (such as nausea, vomiting, etc.) restrict its application. In order to reduce the toxic and side effects of FDU in the treatment of tumor, and to detect the distribution and accumulation of drugs in vivo, this paper uses nitrobenzyl as the hypoxia response group and 2- carboxyl -2 'hydroxybiphenyl (BCM) as the precursor of the fluorescent dye, and coupling the covalent bond with the active drug FDU, and designs and syntheses the anoxy responsive diagnosis and treatment drug FD U-BCM-NO_2. was used to characterize the prodrugs and intermediates by means of NMR, MS and IR, and the HPLC method was used to determine the reductive components of the precursor. The fluorescence intensity of the precursor and Na2S2O4 at different time was measured by the fluorescence spectrum method, and the changes of the release amount of FDU were discussed with the time. FDU and FDU-BCM-NO_2 were tested by MTT method. For the cytotoxicity of MCF-7, MGC-803 tumor cells and BRL-3A normal liver cells, the flow cytometry was used to test the apoptosis of the tumor cells induced by FDU-BCM-NO_2, and the anti tumor effect of FDU-BCM-NO_2 in vitro was evaluated. The hypoxic tumor cells and tumor spheres incubated with FDU-BCM-NO_2 were detected by fluorescence imaging, and the FDU-BCM-NO_2 pair was evaluated. The diagnosis of hypoxia and the tracer effect on FDU showed that, under the hypoxic condition, the active drug FDU and the fluorescent molecule BCM were released while the FDU-BCM-NO_2 was reduced. The release of BCM increased with the prolonged incubation time of hypoxia, and the release of FDU could be monitored by the fluorescence signal of BCM. FDU incubated MCF-7 breast cancer cells in normal oxygen condition, M, respectively, M. The survival rates of GC-803 gastric cancer cells and BRL-3A hepatocytes were 41.43%, 41.70% and 48.09% respectively after 48 h, while the survival rates of three cells incubated in FDU-BCM-NO_2 were 91.09%, 91.22% and 99.08%, respectively, and the survival rates of MCF-7 and MGC-803 incubated by FDU were 22.57% and 41.70%, while FDU-BCM-NO_2 incubation was fine. The cell survival rate was 44.76% and 51.27%, respectively, indicating that FDU had inhibitory effect on normal and tumor cells, while FDU-BCM-NO_2 had hypoxia selectivity, only inhibited hypoxia and did not produce toxicity to normal cells. In hypoxic conditions, FDU-BCM-NO_2 incubated in MGC-803 cells, 52.21% cells were apoptotic, indicating FD U-BCM-NO_2 inhibits the proliferation of tumor cells in the manner of apoptosis, and the release of.FDU-BCM-NO_2 under hypoxic conditions has two photon excitation characteristics. The fluorescence signal can be used to detect the hypoxic and drug release of tumor cells and tumor spheres and to detect the release of drugs. The dual function of.FDU-BCM-NO_2 can be used to realize FD with the hypoxic characteristic of solid tumor. The controlled release of U has avoided its toxic and side effects on normal cells, and provided a new way of thinking for individual treatment of tumors.
【學(xué)位授予單位】：河北大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R914;R96

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