硕士论文--姜黄素类化合物的提取分离鉴定及其与化疗药的协同抗肿瘤作用研究.doc

硕士论文一姜黄素类化合物的提取分离鉴定及其与化疗药的协同抗肿瘤作用研究烟台大学硕士学位论文姜黄素类化合物的提取分离鉴定及其与化疗药的协同抗肿瘤作用研究姓名：田杰申请学位级别:硕士专业:药物化学指导教师:刘珂20090601烟台大学硕士学位论文摘要目的:从中药姜黄中提取分离、鉴定姜黄素类化合物，研究姜黄素单药与临床 常用化疗药联合用药对人纤维肉瘤HT1080细胞、人乳腺癌MCF-7细胞、人 肺腺癌A549细胞的增殖抑制效果及其诱导肿瘤细胞凋亡的协同作用，以探讨其抗 肿瘤的协同作用机制，为临床寻找有效的化疗增效剂提供实验依据和理论基础方法:通过硅胶柱色谱对姜黄80%乙醇提取物进行分离纯化，以理化方法及 波谱学方法对分离得到的化合物进行结构鉴定;采用MTT法评价姜黄素类单体 化合物对人纤维肉瘤HT1080细胞、人乳腺癌MCF-7细胞、人肺腺癌A549细胞 的增殖抑制作用；采用MTT法评价姜黄素单药在HT1080细胞、MCF-7细胞、A549 细胞上与顺伯、阿霉素、羟基喜树碱等药物联合用药的增效作用；通过Hoechst 33258染色观察姜黄素和阿霉素联合用药对A549细胞凋亡的形态学变 化；Western blot法检测对A549细胞Bcl-2蛋白表达的影响。

结果：1分离得到3个姜黄素类化合物，分别鉴定为姜黄素curcumin I,脱甲氧 基姜黄素 demethoxycurcumin II,双脱甲氧基姜黄素 bisdemethoxycurcuminIII；2姜黄素、脱甲氧基姜黄素、双脱甲氧基姜黄素对HT1080细胞、MCF-7细 胞、A549细胞的增殖均有明显的抑制作用，姜黄素的体外增殖抑制效果最好，对三种细胞株的半数抑制浓度IC分别为10.39nmol/L、11.85 umol/L、10. 25U mol/Lo503在一定浓度范围内，姜黄素和顺伯、阿霉素、羟基喜树碱分别联合用药对HT1080细胞、MCF-7细胞、A549细胞的增殖抑制有协同或相加作用4姜黄素和阿霉素联合用药对A549细胞凋亡有协同作用，其作用可能是通 过下调抑凋亡蛋白Be 1-2表达来实现的结论:姜黄素和顺伯、阿霉素、舞基喜树碱分别联合用药对HT1080细胞、MCF-7细胞、A549细胞的增殖抑制有协同或相加作用，其协同抗肿瘤的机制可能与 诱导细胞凋亡,通过下调抑凋亡蛋白Bel-2表达有关体外实验研究表明，姜黄 素与化疗药联合用药对部分肿瘤的治疗具有一定的可行性，在体外抑制人肿瘤细胞 的作I烟台大学硕士学位论文用中有协同增效作用，为临床寻找有效的化疗增效剂提供了一定的实验依据 和理论基础。

关键词:姜黄;姜黄素;脱甲氧基姜黄素;双脱甲氧基姜黄素；增殖抑制；细胞凋亡；AbstractObjective: To extract and isolate curcuminoids from Curcuma longa and study thecurcumin alone combinated with antineoplastic agent in human fibrosarcoma HT1080cell, human breastcancer MCF-7 cell, human lung cancer adenocarcinoma A549 cell onthe proliferation inhibitory effects and apoptosis-induced synergistic effects, which inorder to explore its synergistic anti-tumor mechanisms, the results might be provided anexperimental basis and theoretical foundation for effective chemotherapy synergi stMethod: The 80% EtOII extract was subjected to silica gel column chromatographyChemical structures were identified by physico-chemical properties and spectralanalysis; The effects of curcuminoids on proliferation inhibition of HT1080 cell line,MCF-7 cel 1 line, A549 cel 1 1 ine were detected by MTT assay; The effects of curcuminin combination with cisplatin, adriamycin, hydroxycamptothecin on proliferationinhibition of HT1080 cell line, MCF-7 cell line, A549 cell line were detected by MTTassay; The morphological analysis of A549 cell apoptosis were detected by Hoechst33258 staining; Bcl-2 protein expression in A549 cell were detected by WesternblottingResult: 1 Three curcuminoids were isolated from Curcuma longa and identified ascurcumi n I , demethoxycurcumi n II, bi sdemethoxycurcumi n ITT;2 Curcumin, demethoxycurcumin, bisdemethoxycurcumin al 1 showed significantlyproliferation inhibition on HT1080 cell line, MCF-7 cell line, A549 cell line in vitro,the curcumin indicated the best proliferation inhibition activity, the TC50 wererespectively 10.39 u mol/L, 11. 85 u mol/L and 10.25 u mol/L3 In a certain concentration range, the curcumin in combination with cisplatin,adriamycin, hydroxycamptothecin on HT1080 cell line, MCF-7 cell line, A549 cell1 ine has synergistic or additive effect on proliferation inhibition4 Curcumin combinated with adriamycin has synergistic effect on A549 cell apoptosis,III烟台大学硕士学位论文its mechanisms may be through down-regulating Bcl-2 protein expression to theimplementationConclusion:Curcumin in combination with ci sp1 atin, adriamycin,hydroxycamptothecin on HT1080cell line, MCF-7 cell line, A549 cell line has synergistic or additive effect onproliferation inhibition, the synergistic anti-tumor mechanism may be related toapoptosis-induced and down-regulating Be 1-2 protein expression. The results haveshown that curcumin combined with chemotherapy drugs for the treatment of sometumor may be have the possibi 1 ity, which have synergiStic effect in proliferationinhibition of human tumor cells, it might be provided an experimental basis andtheoretical foundation for effective chemotherapy synergistKey words: Curcuma longa; curcumin; demethoxycurcumi n; bi sdemethoxycurcumin;proliferat ion inhibition; apoptosis;IV烟台大学硕士学位论文中英文名词术语对照姜黄素 curcuminCUR脱甲氧基姜黄素 demethoxycurcumin双脱甲氧基姜黄素bi sdemethoxycurcumin核磁共振XMR薄层层析TLC抗肿瘤 anti-tumor顺铀DDP阿霉素ADM羟基喜树碱HCPT人纤维肉瘤细胞株HT-1080人乳腺癌细胞株MCF-7人肺癌细胞株A549磷酸盐缓冲液PBS二甲基亚W DMSO半数抑制浓度IC50四甲基n塞哇蓝MTT细胞凋亡apoptosis肿瘤坏死因子TNF- a环氧合前COX血管内皮生长因子VEGF三羟甲基氨基甲烷T「is2烟台大学硕士学位论文烟台大学学位论文原创性声明和使用授权说明原创性声明本人郑重声明：所呈交的学位论文，是本人在导师的指导下,独立进行研究工作所取得的成果。

除文中已经注明引用的内容外，本论文不含任何其他个 人或集体已经发表或撰写过的作品或成果对本文的研究做出重要贡献的个人和 集体，均已在文中以明确方式标明本声明的法律结果由本人承担论文作者签名：日期：年月日学位论文使用授权说明本人完全了解烟台大学关于收集、保存、使用学位论文的规定，即：按照学校要求提交学位论文的印刷本和电子版本；学校有权保存学位论文的印刷本和电子版，并提供目录检索与阅览服务；学校可以采用影印、缩印、数字化或其它复制手段保存论文；在不以赢利为目的的前提下，学校可以公布论文的部分或全部内容保密论文在解密后遵守此规定）论文作者签名：导师签名：日期：年月日烟台大学硕士学位论文版权声明任何收存和保管木论文各种版本的单位和个人，未经本论文作者同意，不得将本论文转借他人，亦不得随意复制、抄录、拍照或以任何方式传播否则，引起有碍作者著作权之问题，将可能承担法律责任68烟台大学硕士学位论文第一章综述姜科姜黄素植物姜黄Curcuma Tonga L.是一种常用的中药，收载于历版《中华人民共和国药典》姜黄素是中药材姜黄Rhizoma Curcumae 。