【作者】 孙枝红；

【导师】 王勤；

【作者基本信息】 兰州大学 ， 细胞生物学， 2013， 硕士

【摘要】 Notch信号通路是一条高度保守的信号通路,它参与调控许多重要的生物事件,它在细胞的增殖、分化、凋亡、黏附以及上皮-间质转化等过程中都起着重要作用。如果该信号通路紊乱就可能导致肿瘤的发生。Notch1是Notch家族受体的组成成员之一,在许多癌症中处于异常高表达状态,因此,在这种情况下,可以通过下调Notch1的表达来有效控制Notch信号通路。Hes1是Notch1信号通路下游最为典型的靶基因,在Notch1信号通路中扮演着重要的角色。由于Notch信号通路在乳腺癌发展进程中的重要作用,因此,探寻Notch信号通路抑制剂的一直是乳腺癌研究的热点之一。黄腐酚(Xanthohumol, XN)是发现在啤酒花树脂中的主要的异戊烯基黄酮类化合物,它具有多种生物活性。有文献报道黄腐酚可以抑制乳腺癌细胞的增殖,但其抑制机理不详。本论文主要以Notch信号通路为靶点,探讨黄腐酚抑制乳腺癌细胞生长的作用机理。通过RT-PCR和Western blot检测MCF-7、MDA-MB-231、L02和293T细胞中Notch1和Hes1表达水平,结果表明Notch1和Hes1在MCF-7和MDA-MB-231细胞中高表达；采用双荧光报告系统初步评价黄腐酚对Notch1活性的抑制,Western blot和RT-PCR进一步测定黄腐酚对Notch1和Hes1表达的抑制作用,发现黄腐酚(5、10和20μM)对Notch1和Hes1活性的抑制呈现浓度依赖效应；细胞划痕和Western blot分析表明黄腐酚抑制MDA-MB-231细胞的迁移与Notch1的下调有关；流式细胞周期分析和Western blot结果显示,黄腐酚通过下调Notch1的表达而诱导细胞周期阻滞在G0/G1期；MTT细胞增值和Western blot结果显示黄腐酚可以明显下调Notch1调控的增殖促进基因Survivin和c-myc的表达；Western blot分析表明黄腐酚能够显著下调细胞Notch1调控的抗凋亡基因Bcl-xL和Bcl-2的表达,同时促进凋亡特征性蛋白因子PARP的裂解。综上所述,黄腐酚在MCF-7、MDA-MB-231细胞中通过抑制Notch信号通路中Notch1的表达,从而抑制乳腺癌细胞的生长。提供了黄腐酚可能作为Notch信号通路潜在抑制剂的理论依据,具有良好的应用前景。更多还原

【Abstract】 The Notch signaling pathway is a highly conserved signaling pathway. It is involved in the regulation of many important biological events, including the cell proliferation, differentiation, apoptosis, adhesion, epithelial-mesenchymal transition process. The disorder of Notch signal may lead to tumorigenesis. Notchl is an integral member of the Notch receptor family. In many cancers, the Notchl keeps abnormal high expression, In this case, down-regulating the expression of Notchl can efficiently control the Notch signaling pathway. Hesl is the most typical downstream target gene, which plays an important role in the Notchl signaling pathway. Because of the important role of the Notch signaling pathway in breast cancer development process, the study of the Notch signaling pathway inhibitors has been one of the hotspots of the breast cancer research.Xanthohumol (Xanthohumol, XN) is one of the primary isopentenyl flavonoids found in hops resin. It has a variety of biological activity. Previous studies have reported that Xanthohumol can effectively inhibit breast cancer cell proliferation. However, the mechanisms of Xanthohumol inhibition in breast cancer cell growth remains unclear.In this thesis, we hypothesized that the Notch signaling pathway is a targeted by Xanthohumol leading to inhibit the breast cancer cell growth. In this study, expressions of Notchl and Hesl in MCF-7, MDA-MB-231, L02and293T cells were detected by RT-PCR and Western blot. The results showed that Notch1and Hes1expression were significantly up-regulated in MCF-7and MDA-MB-231cells. Luciferase reporter assays were performed to preliminary assess the function of Xanthohumol inhibition of Notchl activity. The further investigation of Xanthohumol inhibiting Notchl and Hesl expression were performed by Western blot and RT-PCR. The experiment results show that Notchl and Hesl were dose down-regulated by different concentration Xanthohumol treatment (5,10and20μM). Cell wounding heal and Western blot analysis showed that down-regulation of Notchl expression by Xanthohumol treatment inhibited breast cancer cell migration. The flow cytometry cycle analysis and Western blot analysis found that down-regulating expression of Notchl by Xanthohumol treatment induced cell cycle arresting in G0/G1phase. The inhibition of breast cancer cells proliferation by Xanthohumol treatment were measured by MTT assay and Western blot. The results show that Xanthohumol can significantly down-regulate the expression of two proliferation-promoting genes, Survivin and c-myc, which are regulated by Notchl. Western blot analysis showed that Xanthohumol significantly down-regulated the expression of two anti-apoptotic genes, Bcl-xL and Bcl-2, which are also regulated by Notchl. At the same time, Xanthohumol can promote the degradation of PARP, which promotes cell apoptosis. In summary, Xanthohumol can inhibit the Notch1expression of Notch signaling pathway in MCF-7and MDA-MB-231cells, which leads to the inhibition of the breast cancer cell growth. This study provides theory evidence that Xanthohumol can be a potential inhibitor of Notch sinaling pathway. Thus, Xanthohumol has good application prospects.In summary, Xanthohumol is inhibiting the growth of MCF-7and MDA-MB-231cells by inhibiting the the expression of Notchl. The results of these studies provide the theoretical basis of Xanthohumoi may be potential inhibitors of Notch signaling pathway, which may be have a good application prospects.更多还原