本文選題：Rap1 + H-Ras�。� 參考：《哈爾濱工業(yè)大學(xué)》2011年碩士論文

【摘要】：H-Ras和Rap1屬Ras超家族小分子量G蛋白,對(duì)細(xì)胞的增殖、分化、凋亡、細(xì)胞骨架重排等基本生命活動(dòng)起重要的調(diào)控作用。近來(lái)發(fā)現(xiàn)二者在許多細(xì)胞功能上具有拮抗作用,同時(shí)由于二者均存在細(xì)胞膜和細(xì)胞連接位點(diǎn)的定位,且對(duì)細(xì)胞粘附連接具有相互拮抗的調(diào)控作用,因此為探討Rap1和H-Ras在細(xì)胞粘附連接形成中拮抗作用的機(jī)制,本文通過(guò)鈣離子轉(zhuǎn)換實(shí)驗(yàn)(calcium switch experiments)研究了乳腺癌細(xì)胞系MCF7中隨著粘附連接的重建H-Ras和Rap1細(xì)胞內(nèi)定位及相互作用蛋白的改變。 在正常培養(yǎng)條件下GFP-H-Ras、GFP-Rap1以及E-cadherin在MCF7細(xì)胞具有明顯的膜定位,并在細(xì)胞間連接處呈現(xiàn)富集;除此之外GFP-Rap1以及E-cadherin還存在細(xì)胞內(nèi)的定位方式。在低鈣條件下細(xì)胞偽足消失,細(xì)胞間的接觸減少,E-cadherin和GFP-Rap1的膜定位明顯下降,彌散分布于細(xì)胞內(nèi)和質(zhì)膜上;而GFP-H-Ras的細(xì)胞膜定位并沒(méi)有隨著細(xì)胞連接的消失發(fā)生改變。當(dāng)恢復(fù)正常培養(yǎng)條件后,Rap1和E-cadherin均恢復(fù)膜定位,而Rap1要早于E-cadherin恢復(fù),說(shuō)明Rap1可能通過(guò)招募E-cadherin到細(xì)胞膜從而對(duì)依賴于E-cadherin的粘附連接形成有正調(diào)控作用。而H-Ras對(duì)粘附連接的調(diào)控作用可能與E-cadherin的定位無(wú)直接關(guān)系。 同時(shí)我們通過(guò)pull down方法對(duì)在鈣離子轉(zhuǎn)換發(fā)生前后H-Ras與Rap1的共同相互作用蛋白進(jìn)行了分別分離,并分析了它們對(duì)PI3K結(jié)合能力的變化。結(jié)果表明在鈣離子轉(zhuǎn)換后PI3K與H-Ras的結(jié)合減少,而Rap1與PI3K結(jié)合增多。綜上所述,本研究結(jié)果顯示H-Ras和Rap1可能通過(guò)差異調(diào)控E-cadherin的細(xì)胞內(nèi)定位以及與下游信號(hào)分子的結(jié)合來(lái)影響細(xì)胞粘附連接的形成。
[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.
【學(xué)位授予單位】：哈爾濱工業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R329

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 鄭霞;賀愛(ài)蘭;葉啟發(fā);蔣圣軍;;小分子G蛋白R(shí)ap1 shRNA表達(dá)載體的構(gòu)建和鑒定[J];生命科學(xué)研究;2009年02期

2 孫曉杰;黃常志;;PI3K-Akt信號(hào)通路與腫瘤[J];世界華人消化雜志;2006年03期

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本文編號(hào)：1967269