【作者】 张磊；

【导师】 邹伟；

【作者基本信息】 辽宁师范大学 ， 细胞生物学， 2008， 硕士

【摘要】 乳腺癌是女性最常见的恶性肿瘤之一,是乳腺导管上皮细胞在各种内外致癌因素的作用下,失去正常特性而异常增生,以致超过自我修复的限度而发生癌变的疾病。研究表明,许多遗传中与乳腺有关的异常基因和后天受致癌因素影响发生的基因变化参与这一过程。但到目前为止,乳腺上皮细胞恶性转化的分子机制仍不十分清楚,导致乳腺癌发病机理的研究难以取得突破性进展。Caveolin-1(Cav-1,窖蛋白,曾译为小窝蛋白)是Caveolae(胞膜窖)的标志蛋白,主要表达于终末分化的细胞。近年研究发现, Cav-1与乳腺上皮细胞转化和乳腺癌发生密切相关。在我们实验室前期的研究中,利用基因捕获技术得到稳定传代的Cav-1单倍不足(haploinsufficiency)人乳腺上皮细胞株(MCF10A-ST1),Cav-1的mRNA和蛋白质表达均明显降低。进一步研究证实,Cav-1单倍不足可以通过上调乳腺上皮细胞内增殖相关的MAPK信号转导通路等导致人乳腺上皮细胞早期转化,但与Caveolae内其它信号分子的关系尚不完全清楚。钾离子通道(K+通道)是细胞膜上具有转运离子能力的一类重要功能蛋白质,参与细胞许多重要的新陈代谢活动,例如,细胞的增殖、溶质转运、体积控制、酶的激活、细胞分泌、基因表达、兴奋性收缩的传递及细胞内通讯等等。近年来发现,乳腺细胞膜上分布着不同类型的K+通道,其生理机能的改变与乳腺癌的发生发展有着重要的联系。已经证实,多种重要的离子通道定位在Caveolae上,其通道活性受Caveolae的调节。本实验以Cav-1表达水平不同的人乳腺上皮细胞MCF10A、MCF10A-ST1和乳腺癌细胞MCF7为研究对象,首先通过MTT法检测了K+通道阻断剂TEA和4-AP等对三种乳腺细胞和乳腺癌细胞增殖的影响;其次,利用RT-PCR和免疫印迹方法检测了不同Kv通道在三种细胞中的表达水平及免疫共沉淀技术检测了Kv1.5与Cav-1的共表达关系;第三,观察了K+通道阻断剂4-AP作用后对三种细胞中MAPK通路蛋白的影响,旨在探讨K+通道在乳腺细胞早期转化中的作用与Cav-1介导的信号通路的关系,进一步阐明乳腺癌发生、发展的分子机制,为乳腺癌的早期诊断和治疗提供新思路。得到如下结果:1. K+通道的特异性阻断剂TEA和Kv通道阻断剂4-AP均对人乳腺上皮细胞MCF10A、MCF10A-ST1和乳腺癌细胞MCF7的增殖具有抑制作用,且呈剂量时间依赖性。提示乳腺细胞上有大量的K+通道或Kv通道存在,而且K+通道对细胞生长十分重要。2. Kv通道在不同人乳腺上皮细胞中的表达不同,MCF10A细胞中Kv1.5表达水平最高,MCF10A-ST1中表达次之,MCF7细胞中表达最低,而Kv1.2等的表达没有明显差距,这一结果与三种细胞中Cav-1的表达水平一致,且免疫共沉淀显示了Kv1.5与Caveolae具有直接的关系。提示K+通道在乳腺细胞增殖中的作用可能与Cav-1有关。3. Kv通道阻断剂4-AP可能影响MAPK蛋白磷酸化MAPK蛋白。MCF10A、MCF10A-ST1、MCF7细胞单纯4-AP作用对三种细胞p-ERK1/2表达无影响。进一步,4-AP作用后血清刺激,p-ERK1/2表达水平明显增加。结论:1.人乳腺上皮细胞分布不同的Kv通道,影响乳腺细胞的增殖和转化。2.Kv1.5可能参与乳腺细胞的早期转化过程,其表达和功能可能受Cav-1导的信号通路的调节。更多还原

【Abstract】 Breast cancer is the most common kind of malignant tumor and a kind of multi-factor influenced cancer. Both of these factors combine to cause gene mutation or the over-expression of oncogene. Varieties of natural genetic and environmental oncogene are involved in the process. However, the molecular mechanism of malignant transformation for breast epithelial cells is still unclear.Caveolin-1(Cav-1) is the main structural component of non-clathrin, flask-shaped invaginations named Caveolae. Cav-1 is very important in a variety of cellular functions, such as; the endocytic process, homeostasis (both cholesterol and lipid), signal transduction and tumor suppression. Caveolae are thought to serve as sites for the gathering of signaling complexes, thereby facilitating the initiation and cross-talk of signaling events. In previous research, Cav-1 haploinsufficiency cell lines were obtained by using a gene trapping approach , mRNA and protein levels were reduced; furthermore, Cav-1 haploinsufficiencies induce early transformation of human breast epithelial cells by upregulated MAPK signaling pathways. However, the relationship with other signals in Caveolae is still unclear.Potassium channels (K+ channels) are major signaling molecules expressed in a wide range of tissues where they have significant involvement in determining a variety of cellular functions, including; proliferation, solute transport, volume control, enzyme activity, secretion, invasion, gene expression, excitation-contraction coupling and intercellular communication. Previous reports show that K+ channels are linked to the physical activity of breast cancer cells. A current study has confirmed that a variety of important K+ channels localize on the caveolae and are modulated by caveolae.In this study, we used MCF0A, MCF10A-ST1 and MCF7 cells which have expressed different levels of Cav-1. First, an MTT assay was used to observe the inhibitory rate of MCF10A, MCF10A-ST1 and MCF7 cells treated with 4-AP and TEA. Second, RT-PCR and western blot techniques were used to analyze the expression of K+ channels. Co-immunoprecipitation was then used to investigate the link between K+ and Caveolin-1. Third, the western blot was again used to detect MAPK signal pathways of MCF10A, MCF10A-ST1, MCF7 cells after being treated with 4-AP. The object of this study was to investigate the effect of K+ channels during the transformation of breast cells and the mechanism of signaling pathways associated with Cav-1; while the aim was to investigate the mechanism of breast cancer’s occurrence and development. Furthermore, another aim was to provide a new method for diagnosis and therapy in breast cancer.Results:1. The effects of TEA (K+ channel blocker) and 4-AP (Kv channel blocker) on proliferation of the human breast epithelial cells MCF10A, MCF-10A-ST1 and breast cancer cells MCF7 are dose-time-dependent. Results indicated that many K+ channels and Kv channels distribute on breast cell membranes and they are important in cell growth.2. The expression of Kv channels of breast cells are different. Expression of Kv1.5, Kv1.2 in MCF10A, MCF10A-ST1, MCF7 indicates that expression of Kv 1.5 (but not Kv1.2) is reduced with breast cancer progression. Expression of Cav-1 is reduced with breast cancer progression and immuno-precipitation studies show that the Kv1.5 is associated with caveolin-1. It suggested that effect of K+ channels in the proliferation of breast cells relate to the role of Cav -1.3. p-ERK1/2 protein level was not effected by incubation with 4-AP (no serum) after serum starvation. However, p-ERK1 /2 protein levels were increased significantly after incubation with 4-AP (added serum after 4-AP 10min). It indicated that 4-AP might effect the phosphorylation of MAPK in human breast epithelial cells.Conclusion:1. The present results demonstrated that a variety of Kv distribute on the breast cell membrane and Kv channels play important role during the transformation and proliferation of human breast epithelial cells.2. We hypothesize that Kv1.5 might be involved in the early transformation of breast epithelial cells. Its activity and expression were modulated by the signal pathway associated Cav-1.更多还原