Rap1及Ras在细胞粘附连接中的功能研究

发布时间：2018-06-02 03:55

本文选题：Rap1 + H-Ras　；参考：《哈尔滨工业大学》2011年硕士论文

【摘要】：H-Ras和Rap1属Ras超家族小分子量G蛋白,对细胞的增殖、分化、凋亡、细胞骨架重排等基本生命活动起重要的调控作用。近来发现二者在许多细胞功能上具有拮抗作用,同时由于二者均存在细胞膜和细胞连接位点的定位,且对细胞粘附连接具有相互拮抗的调控作用,因此为探讨Rap1和H-Ras在细胞粘附连接形成中拮抗作用的机制,本文通过钙离子转换实验(calcium switch experiments)研究了乳腺癌细胞系MCF7中随着粘附连接的重建H-Ras和Rap1细胞内定位及相互作用蛋白的改变。在正常培养条件下GFP-H-Ras、GFP-Rap1以及E-cadherin在MCF7细胞具有明显的膜定位,并在细胞间连接处呈现富集;除此之外GFP-Rap1以及E-cadherin还存在细胞内的定位方式。在低钙条件下细胞伪足消失,细胞间的接触减少,E-cadherin和GFP-Rap1的膜定位明显下降,弥散分布于细胞内和质膜上;而GFP-H-Ras的细胞膜定位并没有随着细胞连接的消失发生改变。当恢复正常培养条件后,Rap1和E-cadherin均恢复膜定位,而Rap1要早于E-cadherin恢复,说明Rap1可能通过招募E-cadherin到细胞膜从而对依赖于E-cadherin的粘附连接形成有正调控作用。而H-Ras对粘附连接的调控作用可能与E-cadherin的定位无直接关系。同时我们通过pull down方法对在钙离子转换发生前后H-Ras与Rap1的共同相互作用蛋白进行了分别分离,并分析了它们对PI3K结合能力的变化。结果表明在钙离子转换后PI3K与H-Ras的结合减少,而Rap1与PI3K结合增多。综上所述,本研究结果显示H-Ras和Rap1可能通过差异调控E-cadherin的细胞内定位以及与下游信号分子的结合来影响细胞粘附连接的形成。
[Abstract]:H-Ras and Rap1 are small molecular weight G proteins of Ras superfamily, which play an important role in regulating cell proliferation, differentiation, apoptosis and cytoskeleton rearrangement. Recently, two have been found to have antagonistic effects on many cell functions. At the same time, the location of cell membrane and cell junction sites in both of the two groups and the adhesion and attachment of cells to cells are also found. In order to explore the antagonistic role of antagonism, so as to explore the mechanism of antagonism between Rap1 and H-Ras in cell adhesion formation, this paper studied the localization of H-Ras and Rap1 in the cell line MCF7 of breast cancer cell line MCF7, and the changes in the interaction proteins in the MCF7 cell line of the breast cancer cell line (calcium switch experiments).
Under normal culture conditions, GFP-H-Ras, GFP-Rap1 and E-cadherin have obvious membrane location in MCF7 cells, and are enriched at the intercellular junction. In addition, GFP-Rap1 and E-cadherin also exist in the cell location mode. Under low calcium conditions, the cell hypoplastic foot disappears, the contact between cells decrease, and the membrane location of E-cadherin and GFP-Rap1 is located. The location of GFP-H-Ras's cell membrane did not change with the disappearance of the cell connection. When the normal culture conditions were restored, both Rap1 and E-cadherin restored the location of the membrane, while Rap1 was earlier than E-cadherin, indicating that Rap1 may be dependent on E- by recruiting E-cadherin to the cell membrane. There is a positive regulatory effect on the adhesion and adhesion of cadherin, while the regulation of H-Ras on adhesion junction may not be directly related to the location of E-cadherin.
At the same time, the pull down method was used to separate the common interacting proteins between H-Ras and Rap1 before and after the calcium conversion, and the changes in the binding capacity of PI3K were analyzed. The results showed that the binding of PI3K to H-Ras decreased after the calcium ion conversion, and the combination of Rap1 and PI3K increased. In summary, the results of this study show H-R. As and Rap1 may influence the formation of cell adhesion and adhesion by differentiating the intracellular localization of E-cadherin and binding with downstream signaling molecules.
【学位授予单位】：哈尔滨工业大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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