对叶大戟大环二萜类化合物ES2逆转肿瘤多药耐药的实验研究

发布时间：2018-03-09 12:45

本文选题：对叶大戟　切入点：肿瘤多药耐药　出处：《华东师范大学》2016年硕士论文　论文类型：学位论文

【摘要】：背景：肿瘤多药耐药(multidrug resistance, MDR)是肿瘤化疗治疗失败的重要原因之一,至今尚未有任何一种MDR逆转剂获批应用于临床治疗。传统的中草药凭借其化学多样性、毒副作用小、成本低等特点逐渐成为科学家寻找MDR逆转剂的重要来源。中国科学院新疆理化技术研究所阿吉艾克拜尔·艾萨研究员课题组首次从大戟属对叶大戟中分离得到9个结构新颖的假白榄烷型大环二萜类化合物,生物学活性筛选结果表明,这些化合物对肿瘤细胞的生长抑制作用均较弱,且普遍具有一定的肿瘤多药耐药逆转活性。目的：本实验旨在对阿吉艾克拜尔·艾萨研究员课题组从大戟属对叶大戟、粗根大戟和准噶尔大戟中分离得到的54个萜类天然化合物进行抗肿瘤活性、抗耐药活性和耐药逆转活性的评价和筛选,以期发现具有显著的肿瘤多药耐药逆转活性的候选化合物,并采用肿瘤药理学、细胞生物学和分子生物学的方法和技术对该候选化合物进行系统的药理、药效学评价,以及对其耐药逆转作用机制进行深入探讨,以寻找具有新颖结构的Pgp抑制剂先导化合物,为开发具有自主知识产权的肿瘤MDR逆转剂奠定早期新药研发基础。方法：1.采用MTT法、流式细胞术对从粗根大戟、准噶尔大戟和对叶大戟中分离得到的54个萜类化合物进行生物活性筛选,评价得到耐药逆转活性最强的化合物ES2。2.MTT法检测ES2对肿瘤细胞亲本株和耐药株的生长抑制作用、抗耐药作用及耐药逆转作用。3. 荧光显微观察和流式细胞术检测ES2对肿瘤细胞亲本株及耐药株中Rho123或DOX的蓄积及外排的影响。4. Pgp-GloTM Assay Systems检测ES2对Pgp ATP酶活力的影响。5. Docking实验分析ES2与Pgp、维拉帕米的相互作用。6.蛋白免疫印迹实验检测ES2对耐药株细胞内Pgp表达的影响及具体机制。7.实时荧光定量PCR技术检测ES2对KBv200细胞内Pgp mRNA表达水平的影响。8.MTT法检测ES2耐药逆转活性的持续性。结果：1.在从新疆维吾尔族中草药大戟属植物中提取得到的54个萜类天然化合物中,对叶大戟假白榄烷型大环二萜类化合物ES1-7,粗根大戟二萜类化合物EM-23和准噶尔大戟萜类化合物E13,E14和E18等新结构化合物对肿瘤细胞亲本株和耐药株的生长抑制作用相当,表现出一定的抗肿瘤耐药作用,并显著增加耐药株KBv200细胞内Rho123的蓄积量,其中对叶大戟假白榄烷型大环二萜类化合物ES2的活性最强。2.ES2对三对肿瘤细胞亲本株和耐药株(KB、KBv200、MCF-7、MCF-7/ADR、A549和A549T)生长抑制作用相当,活性较弱(ICs030μM),表现出一定的抗肿瘤耐药作用。但具有很强的逆转肿瘤MDR活性,呈浓度依赖性增强传统抗肿瘤药(NVB、PTX和DOX)对耐药株细胞(KBv200和MCF-7/ADR)的细胞毒作用,3 gM时的作用效果已与10μM的阳性药维拉帕米相当,但对亲本株细胞无影响。3.ES2呈浓度依赖方式增强耐药株细胞KBv200和MCF-7/ADR中Rho123或DOX的蓄积,且3 gM时的作用效果已优于10μM的阳性药维拉帕米。此外,ES2还显著抑制KBv200细胞内Rho123的外排。4.ES2呈浓度依赖式地增强了Pgp ATP酶活力,在3.125μM时酶活力达到最大值,在3.125-50μM时酶活力几乎保持不变,且不同浓度的ES2并不影响维拉帕米(VPL)刺激的ATP酶活力。5.ES2和VPL竞争性地结合于Pgp,但结合位点不完全重合。6.ES2下调耐药株细胞中Pgp的蛋白表达水平,但不影响Pgp mRNA表达水平。7.ES2诱导的Pgp下调可能与泛素-蛋白酶体降解途径相关,但可能与自噬无关。8.ES2的耐药逆转活性有一定的可持续性。结论：ES2通过下调耐药株细胞中的Pgp蛋白表达、增强Pgp ATP酶活力并可能作为底物竞争性抑制Pgp对传统抗肿瘤药物的外排,增加其在耐药肿瘤细胞中的蓄积,进而发挥逆转肿瘤MDR活性,是一个非常有潜力的肿瘤MDR逆转先导化合物,为开发具有自主知识产权的具有新颖结构的肿瘤MDR逆转剂奠定了早期药物研发基础。
[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.

【学位授予单位】：华东师范大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R285

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