本文選題：對(duì)葉大戟　切入點(diǎn)：腫瘤多藥耐藥　出處：《華東師范大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

【摘要】：背景：腫瘤多藥耐藥(multidrug resistance, MDR)是腫瘤化療治療失敗的重要原因之一,至今尚未有任何一種MDR逆轉(zhuǎn)劑獲批應(yīng)用于臨床治療。傳統(tǒng)的中草藥憑借其化學(xué)多樣性、毒副作用小、成本低等特點(diǎn)逐漸成為科學(xué)家尋找MDR逆轉(zhuǎn)劑的重要來源。中國科學(xué)院新疆理化技術(shù)研究所阿吉艾克拜爾·艾薩研究員課題組首次從大戟屬對(duì)葉大戟中分離得到9個(gè)結(jié)構(gòu)新穎的假白欖烷型大環(huán)二萜類化合物,生物學(xué)活性篩選結(jié)果表明,這些化合物對(duì)腫瘤細(xì)胞的生長抑制作用均較弱,且普遍具有一定的腫瘤多藥耐藥逆轉(zhuǎn)活性。目的：本實(shí)驗(yàn)旨在對(duì)阿吉艾克拜爾·艾薩研究員課題組從大戟屬對(duì)葉大戟、粗根大戟和準(zhǔn)噶爾大戟中分離得到的54個(gè)萜類天然化合物進(jìn)行抗腫瘤活性、抗耐藥活性和耐藥逆轉(zhuǎn)活性的評(píng)價(jià)和篩選,以期發(fā)現(xiàn)具有顯著的腫瘤多藥耐藥逆轉(zhuǎn)活性的候選化合物,并采用腫瘤藥理學(xué)、細(xì)胞生物學(xué)和分子生物學(xué)的方法和技術(shù)對(duì)該候選化合物進(jìn)行系統(tǒng)的藥理、藥效學(xué)評(píng)價(jià),以及對(duì)其耐藥逆轉(zhuǎn)作用機(jī)制進(jìn)行深入探討,以尋找具有新穎結(jié)構(gòu)的Pgp抑制劑先導(dǎo)化合物,為開發(fā)具有自主知識(shí)產(chǎn)權(quán)的腫瘤MDR逆轉(zhuǎn)劑奠定早期新藥研發(fā)基礎(chǔ)。方法：1.采用MTT法、流式細(xì)胞術(shù)對(duì)從粗根大戟、準(zhǔn)噶爾大戟和對(duì)葉大戟中分離得到的54個(gè)萜類化合物進(jìn)行生物活性篩選,評(píng)價(jià)得到耐藥逆轉(zhuǎn)活性最強(qiáng)的化合物ES2。2.MTT法檢測ES2對(duì)腫瘤細(xì)胞親本株和耐藥株的生長抑制作用、抗耐藥作用及耐藥逆轉(zhuǎn)作用。3. 熒光顯微觀察和流式細(xì)胞術(shù)檢測ES2對(duì)腫瘤細(xì)胞親本株及耐藥株中Rho123或DOX的蓄積及外排的影響。4. Pgp-GloTM Assay Systems檢測ES2對(duì)Pgp ATP酶活力的影響。5. Docking實(shí)驗(yàn)分析ES2與Pgp、維拉帕米的相互作用。6.蛋白免疫印跡實(shí)驗(yàn)檢測ES2對(duì)耐藥株細(xì)胞內(nèi)Pgp表達(dá)的影響及具體機(jī)制。7.實(shí)時(shí)熒光定量PCR技術(shù)檢測ES2對(duì)KBv200細(xì)胞內(nèi)Pgp mRNA表達(dá)水平的影響。8.MTT法檢測ES2耐藥逆轉(zhuǎn)活性的持續(xù)性。結(jié)果：1.在從新疆維吾爾族中草藥大戟屬植物中提取得到的54個(gè)萜類天然化合物中,對(duì)葉大戟假白欖烷型大環(huán)二萜類化合物ES1-7,粗根大戟二萜類化合物EM-23和準(zhǔn)噶爾大戟萜類化合物E13,E14和E18等新結(jié)構(gòu)化合物對(duì)腫瘤細(xì)胞親本株和耐藥株的生長抑制作用相當(dāng),表現(xiàn)出一定的抗腫瘤耐藥作用,并顯著增加耐藥株KBv200細(xì)胞內(nèi)Rho123的蓄積量,其中對(duì)葉大戟假白欖烷型大環(huán)二萜類化合物ES2的活性最強(qiáng)。2.ES2對(duì)三對(duì)腫瘤細(xì)胞親本株和耐藥株(KB、KBv200、MCF-7、MCF-7/ADR、A549和A549T)生長抑制作用相當(dāng),活性較弱(ICs030μM),表現(xiàn)出一定的抗腫瘤耐藥作用。但具有很強(qiáng)的逆轉(zhuǎn)腫瘤MDR活性,呈濃度依賴性增強(qiáng)傳統(tǒng)抗腫瘤藥(NVB、PTX和DOX)對(duì)耐藥株細(xì)胞(KBv200和MCF-7/ADR)的細(xì)胞毒作用,3 gM時(shí)的作用效果已與10μM的陽性藥維拉帕米相當(dāng),但對(duì)親本株細(xì)胞無影響。3.ES2呈濃度依賴方式增強(qiáng)耐藥株細(xì)胞KBv200和MCF-7/ADR中Rho123或DOX的蓄積,且3 gM時(shí)的作用效果已優(yōu)于10μM的陽性藥維拉帕米。此外,ES2還顯著抑制KBv200細(xì)胞內(nèi)Rho123的外排。4.ES2呈濃度依賴式地增強(qiáng)了Pgp ATP酶活力,在3.125μM時(shí)酶活力達(dá)到最大值,在3.125-50μM時(shí)酶活力幾乎保持不變,且不同濃度的ES2并不影響維拉帕米(VPL)刺激的ATP酶活力。5.ES2和VPL競爭性地結(jié)合于Pgp,但結(jié)合位點(diǎn)不完全重合。6.ES2下調(diào)耐藥株細(xì)胞中Pgp的蛋白表達(dá)水平,但不影響Pgp mRNA表達(dá)水平。7.ES2誘導(dǎo)的Pgp下調(diào)可能與泛素-蛋白酶體降解途徑相關(guān),但可能與自噬無關(guān)。8.ES2的耐藥逆轉(zhuǎn)活性有一定的可持續(xù)性。結(jié)論：ES2通過下調(diào)耐藥株細(xì)胞中的Pgp蛋白表達(dá)、增強(qiáng)Pgp ATP酶活力并可能作為底物競爭性抑制Pgp對(duì)傳統(tǒng)抗腫瘤藥物的外排,增加其在耐藥腫瘤細(xì)胞中的蓄積,進(jìn)而發(fā)揮逆轉(zhuǎn)腫瘤MDR活性,是一個(gè)非常有潛力的腫瘤MDR逆轉(zhuǎn)先導(dǎo)化合物,為開發(fā)具有自主知識(shí)產(chǎn)權(quán)的具有新穎結(jié)構(gòu)的腫瘤MDR逆轉(zhuǎn)劑奠定了早期藥物研發(fā)基礎(chǔ)。
[Abstract]:Background: tumor multidrug resistance (multidrug resistance MDR) is one of the important reasons for the failure of tumor chemotherapy, there has not been any kind of MDR reversal agents approved for use in clinical treatment. Traditional Chinese herbal medicine with its chemical diversity, side effects of small, low cost has become an important source of scientists looking for MDR reversal agents Chinese. Xinjiang Academy of physical and chemical research group, researcher Akbar Esa Institute of technology is for the first time from Euphorbia Euphorbia sororia isolated 9 novel fake white elemane macrocyclic two terpenoids, biological activity screening showed that these compounds were weak inhibitory effect on the growth of tumor cells, and generally have a certain the MDR reversal activity. Objective: This study was designed to research group researcher Agiakbar Esa from Euphorbia Euphorbiaceae, coarse root large 54 terpenoid natural compounds isolated from the Junggar Euphorbia halberd and antitumor activity against resistant evaluation activity and resistance reversal activity and screening, found that the candidate compounds have significant MDR reversal activity in order, and the tumor pharmacology, method and technology of cell biology and molecular biology system the pharmacology of this candidate compound, pharmacodynamic evaluation, and the reversal mechanism are discussed, with Pgp inhibitor compounds with novel structure to find tumor MDR reversal agent is developed with independent intellectual property rights for early drug development. Methods: 1. by MTT method, flow cytometry from Euphorbia coarse root, 54 terpene compounds isolated from Euphorbia Euphorbiaceae in Junggar and biological activity screening, evaluation to reverse the resistance of the most active compounds Detection of inhibitory effect of ES2 ES2.2.MTT on tumor cells of parental and resistant strains of growth, analysis of ES2 and anti Pgp effect of.4. Pgp-GloTM Assay Systems ES2 to detect the reversal effect and resistant.3. fluorescence microscopy and flow cytometry ES2 on tumor cells and the parental strain Rho123 or DOX resistant strains of accumulation and efflux the Pgp activity of ATP.5. Docking experiment, continuous interaction of.6. by Western blot detection of ES2 Vera Pammy's influence on the expression of Pgp in cells of the resistant strains and the specific mechanism of.7. real-time fluorescence quantitative PCR detection of ES2 on the expression of Pgp mRNA.8.MTT assay in KBv200 cells to detect ES2 levels of drug resistance reversal activity. Results: 54 terpenoid natural compounds in 1. from Xinjiang Uygur herbs Euphorbia extracted, Euphorbia sororia false white elemane macrocyclic two terpene compounds ES1-7, coarse root Euphorbia two terpenoids EM-23 and Junggar Euphorbia diterpenoid compound E13, E14 and E18 and other new compounds on tumor cells in parental and resistant strains showed growth inhibition, anti tumor drug resistance effect, and significantly increase the volume of drug resistant strains in KBv200 cells Rho123, the leaf a fake white elemane type two macrocyclic diterpenoid compound ES2 of.2.ES2 was the strongest in three of tumor cell parent and resistant strains (KB, KBv200, MCF-7, MCF-7/ADR, A549 and A549T) growth inhibition, weak activity (ICs030, M) showed antitumor drug resistance but has effect. A strong reversal of tumor activity of MDR showed a concentration dependent enhancement of traditional antitumor drugs (NVB, PTX and DOX) of resistant strains (KBv200 and MCF-7/ADR) cell cytotoxicity, positive drug Vera Pammy effect at 3 gM and 10 M have been quite, but for Parental cells had no effect on the concentration of.3.ES2 enhanced the accumulation of Rho123 or DOX cells KBv200 and MCF-7/ADR resistant strains, the positive drug Vera Pammy and the effect of 3 gM is better than 10 M. In addition, ES2 also inhibited KBv200 cells Rho123 efflux.4.ES2 showed a concentration dependent enhancement in Pgp type ATP enzyme activity, enzyme activity reached 3.125 M when the maximum value in the 3.125-50, M enzyme activity remained almost unchanged, and different concentrations of ES2 did not affect Vera Pammy (VPL) stimulation of the enzyme activity of ATP.5.ES2 and VPL competitively bind to Pgp binding sites, but not completely overlap the down-regulation of.6.ES2 resistant strains in cells the protein expression level of Pgp, but does not affect the Pgp expression level of mRNA.7.ES2 induced down-regulation of Pgp may be associated with the ubiquitin proteasome degradation pathway, drug resistance reversal activity but may have unrelated to.8.ES2 and autophagy in certain sustainability. Conclusion ES2 resistant strains in cells through down-regulation of Pgp protein expression, enhanced Pgp ATP activity and may act as a substrate competitive inhibition of Pgp on the traditional antitumor drug efflux, increase the resistance of tumor cells in the accumulation, and then play the activity of MDR reverse tumor is a potential tumor MDR reversal lead compounds for the development, with independent intellectual property rights with tumor MDR reversal agents with novel structure laid the early foundation for drug research and development.

【學(xué)位授予單位】：華東師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R285

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