【作者】 陈馨；

【导师】 姚群峰；

【作者基本信息】 湖北中医药大学 ， 临床检验诊断学， 2010， 硕士

【摘要】 目的：姜黄素(curcumin)是从我国传统中药姜黄根茎中提取的一种植物多酚。现代药理学研究表明,姜黄素具有抗氧化、抗突变、抗肿瘤等多方面的药理作用,体外实验及动物实验证实,姜黄素可以在多个靶点抑制肿瘤细胞的生长和繁殖,抑制大鼠皮肤癌、胃癌、十二指肠癌、结肠癌及乳腺癌等的发生。目前研究认为其抗肿瘤可能涉及诱发肿瘤细胞凋亡阻断肿瘤细胞信号传导通路、抗氧化和抑制肿瘤血管生成等方面。但由于肿瘤的发生是一个多因素、多基因相关的复杂过程,姜黄素的抗肿瘤机制研究有待进一步深入。近年来,表遗传(epigenetic)机制在肿瘤发生中的作用正引起高度关注,它是发生在基因表达水平的可遗传的、无DNA序列的变化。DNA甲基化就是一种与肿瘤形成密切相关的表遗传机制,研究显示,DNA甲基化异常可引起基因表达水平的明显改变,是细胞癌变过程中的一个早期事件。同时,DNA甲基化也是一个可逆的过程,因此此过程有可能成为很好的药物干预靶点。有报道初步显示姜黄素可能是潜在的甲基化抑制剂,本文从DNA甲基化角度探讨姜黄素抗肿瘤的表遗传机制,研究了不同浓度的姜黄素对体外培养的肿瘤细胞的生长抑制作用及对p16基因和MGMT基因甲基化状态的影响,为姜黄素等中药成分的抗肿瘤机理研究提供一个新的途径,同时为姜黄素的临床应用提供科学的理论依据。方法：1.将人胃癌细胞株SGC-7901,人宫颈癌细胞株HeLa,人肝癌细胞株HepG2在体外培养至对数生长期,加入不同浓度的姜黄素(20、40、60μmol/L)分别处理三种肿瘤细胞48小时。在倒置显微镜观察不同浓度姜黄素处理的SGC-7901细胞、HeLa细胞、HepG2细胞后发生的形态学改变；运用基于酶标仪的MTT方法检测肿瘤细胞的增殖情况。2.利用甲基化特异性PCR (Methylation-specific PCR, MSP)方法检测不同剂量的姜黄素作用前后三种肿瘤细胞中p16基因和MGMT基因甲基化状态的改变。3. RT-PCR法检测三种肿瘤细胞经姜黄素处理前后其p16基因和MGMT基因表达水平。结果：1.姜黄素能够抑制SGC-7901细胞、HeLa细胞、HepG2细胞的生长与增殖,并诱导三种肿瘤细胞发生凋亡。2. SGC-7901细胞、HeLa细胞、HepG2细胞p16基因和MGMT基因的启动子区CpG岛呈异常甲基化状态。用姜黄素处理后,低浓度(20μmol/L)的姜黄素对三种肿瘤细胞p16基因和MGMT基因无明显的去甲基化作用,中浓度40μmol/L与高浓度(60μmol/L)姜黄素处理后的SGC-7901细胞、HeLa细胞、HepG2细胞,p16基因和MGMT基因的启动子区CpG岛甲基化程度下降。3.经姜黄素处理后,HeLa细胞p16基因和SGC-7901细胞的MGMT基因重新表达或表达增强,且呈一定的剂量依赖性。结论：姜黄素对SGC-7901细胞、HeLa细胞、HepG2细胞的增殖具有明显抑制作用,且表现出剂量依赖性(P<0.01)；中浓度和高浓度的姜黄素溶液对三种肿瘤细胞p16基因和MGMT基因具有一定的去甲基化作用。姜黄素可使肿瘤细胞中的p16基因和MGMT基因重新表达及表达增强。本研究显示姜黄素对甲基化异常的抑制作用是其一种可能的抗肿瘤机制,为姜黄素应用于肿瘤临床提供了理论依据。更多还原

【Abstract】 Objective:Curcumin is one of the plant polyphenol from turmeric rhizome of traditional Chinese medicine. Modern pharmacology studies have shown that curcumin has anti-oxidation, anti-mutagenic, anti-tumor, and many other pharmacological effects.In vitro and animal experiments demonstrated that curcumin can inhibit tumor cells growth and reproduction in multiple target, restrain skin cancer, stomach cancer, duodenal cancer, colon cancer and breast cancer and other place. Current research suggest that its anti-tumor may be involved induce tumor cell apoptosis, block tumor cell signaling pathways, anti-oxidation and inhibition of tumor angiogenesis and so on. However, the incidence of cancer is a multi-factor, multi-gene-related complex process.The further studies of anti-tumor mechanism of curcumin is necessory.In recent years, epigenetic mechanism in role of tumor is causing great concern. It is happened in gene expression level of genetic, without change of DNA sequences. DNA methylation is a epigenetic mechanisms which is closely related with tumor formation. studies have shown that DNA abnormal methylation can cause significant changes in gene expression,and it is an early event in the process of cancerous cells. At the same time, the DNA methylation is a reversible process, and it may become a good target for drug intervention. Preliminary reports show that curcumin is a potential methylation inhibitor.In our research, the epigenetic mechanism of curcumin anti-tumor was studied. The growth inhibition and the methylation status effect of p16 and MGMT gene of cultured cancer cell treated by the different concentrations of curcumin were detected. A new way was provided for the clinical application research of the Chinese native medicine ingredient,and the results can be the theoretical basis for the clinical application of curcuminMethods:1. In vitro, SGC-7901, HeLa and HepG2 cells were induced logarithmic growth phase, by adding different concentrations of curcumin (20,40,60μmol /L) be dealt with separately three kinds of tumor cells for 48 hours. Use the inverted microscope observation the morphological changes before and after the effects of different concentrations of curcumin treated SGC-7901 cells, HeLa cells, HepG2 cells. Based on the MTT microplate to detect the proliferation of tumor cells.2. Using methylation-specific PCR (MSP) of different doses of curcumin were detected before and after three kinds of tumor cells in the p16 gene and MGMT gene methylation status changes.3. To use RT-PCR detected three kinds of tumor cells by curcumin treatment before and after the p16 gene and MGMT gene expression level.Results:1. Curcumin can inhibit SGC-7901,HeLa and HepG2 cell growth and proliferation and induce apoptosis.2. Treated by curcumin, the p16 gene and MGMT gene the promoter region of CpG island of the SGC-7901, HeLa and HepG2 cells showed abnormal methylation status.Low concentration (20μmol/L) of curcumin treated, p16 gene and MGMT gene have no demethylation. Treated with 40μmol/L and high concentration (60μmol/L) curcumin,the p16 gene and MGMT gene promoter region CpG island was hypermethylation.3. After treated by curcumin, the p16 gene of HeLa cells and the MGMT gene of SGC-7901 cells re-expressed or increased, and showed a dose-dependent.Conclusion:Curcumin significantly inhibits SGC-7901,HeLa and HepG2 cell proliferation, and showed a dose-dependent manner (P<0.01).middle and high concentration of curcumin in three kinds of tumor cells, p16 and MGMT gene has some role in demethylation. Curcumin can cause the p16 gene and the MGMT gene in tumor cells re-expression and expression increased. Our research shows that curcumin can inhibition the abnormal methylation which it may be one of the anti-tumor mechanism, and provide a theoretical basis for the clinical tumor therapy.更多还原