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  本文選題：PP1α + PP1γ��； 參考：《湖南師范大學(xué)》2011年碩士論文

【摘要】：蛋白質(zhì)的磷酸化與去磷酸化過程是生物體內(nèi)一種普遍存在的調(diào)節(jié)方式,幾乎涉及所有的生理及病理過程,并且在細胞信號的傳遞過程中占有極其重要的位置。 蛋白質(zhì)的磷酸化和去磷酸化反應(yīng)分別由蛋白激酶和蛋白磷酸酶催化。蛋白激酶由一個龐大的并且功能極其多樣的基因家族所構(gòu)成,相比之下,蛋白磷酸酶則有著較少的數(shù)量和種類。蛋白磷酸酶1(PP1)的催化亞基能與大于150種的調(diào)節(jié)亞基形成復(fù)合物,這種復(fù)合物的構(gòu)造使得PP1c轉(zhuǎn)變成很多不同的形態(tài),從而具有底物特異性、獨特的亞細胞定位和特異性的調(diào)節(jié)功能。這使得大量的依靠PPl的細胞功能通過獨立的機制得到控制。 我們以H1299為實驗材料,通過運用脂質(zhì)體轉(zhuǎn)染的方法,建立了H1299-pCI-neo、H1299-pCI-PP1α、H1299-pLKO.1和H1299-pLKO.1-PP1α等穩(wěn)定表達細胞株,通過Western Blot的方法鑒定和檢測了PP1α和PP1γ的相對表達情況,我們發(fā)現(xiàn)：當(dāng)過表達PP1α?xí)r,H1299細胞中的PP1γ的表達量出現(xiàn)了明顯的下調(diào),而當(dāng)靶向沉默PP1α?xí)r,H1299細胞中的PP1γ表達水平則出現(xiàn)了明顯的上調(diào),并且,PP1β和PP1γ的表達似乎維持著總量的平衡,二者呈現(xiàn)出拮抗表達的關(guān)系。通過RT-PCR,我們驗證了在mRNA水平也同樣存在著這種關(guān)系,這說明PP1α和PP1γ拮抗表達的關(guān)系在mRNA水平就受到了嚴格地調(diào)控。 我們還通過細胞增殖實驗和裸鼠荷瘤實驗,對H1299、H1299-pCI-neo、H1299-pCI-PP1α三種穩(wěn)定細胞株進行了鑒定,發(fā)現(xiàn)過表達PP1α能明顯的延緩細胞周期并抑制腫瘤細胞的生長,同時,我們也通過Western Blot檢測了幾種基本的周期蛋白的表達,發(fā)現(xiàn)CyclinA/B/E與PP1α的表達具有協(xié)同效應(yīng),而其具體的作用機制有待進一步研究驗證。 綜上,我們可以得出以下結(jié)論：在H1299細胞中,過表達PP1α后PP1γ的表達明顯下調(diào),相反靶向沉默PP1α則PP1γ的表達明顯上調(diào),二者的表達具有拮抗關(guān)系,并維持著總量的穩(wěn)定。同時過表達PP1α能夠阻抑H1299細胞的生長周期的進行,具有良好的抑瘤作用效果。
[Abstract]:The process of protein phosphorylation and dephosphorylation is a universal regulation mode in vivo, which involves almost all physiological and pathological processes, and plays an extremely important role in the process of cell signal transmission. Protein phosphorylation and dephosphorylation are catalyzed by protein kinase and protein phosphatase respectively. Protein kinases consist of a large and highly functional family of genes, whereas protein phosphatase has a smaller number and variety. The catalytic subunit of protein phosphatase 1 (PP1) can form complexes with more than 150 regulatory subunits, which are constructed to transform PP1c into many different forms, thus making it substrate-specific. Unique subcellular localization and specific regulatory functions. This allows a large number of PPl dependent cell functions through independent mechanisms to be controlled. Using H1299 as the experimental material, the stable expression cell lines H1299-pCI-PP1 偽, H1299-pLKO.1 and H1299-pLKO.1-PP1 偽 were established by using liposome transfection. The relative expression of PP1 偽 and PP1 緯 was identified and detected by Western Blot. We found that the expression of PP1 緯 in H1299 cells was significantly down-regulated when PP1 偽 was over-expressed, while the expression level of PP1 緯 in H1299 cells was up-regulated when PP1 偽 was silenced, and the expression of PP1 尾 and PP1 緯 seemed to maintain a balance. There was an antagonistic relationship between them. By RT-PCR, we verified that the same relationship exists at the mRNA level, which indicates that the antagonistic expression of PP1 偽 and PP1 緯 is strictly regulated at the mRNA level. We also identified three stable cell lines H1299-pCI-neoH1299-pCI-PP1 偽 by cell proliferation test and tumor-bearing assay in nude mice. It was found that overexpression of PP1 偽 could significantly delay cell cycle and inhibit the growth of tumor cells. We also detected the expression of several basic cyclins by Western Blot. We found that the expression of CyclinA/B/E and PP1 偽 has synergistic effect, and its specific mechanism needs to be further studied and verified. In conclusion, we can draw the following conclusions: in H1299 cells, the expression of PP1 緯 is down-regulated after overexpression of PP1 偽, whereas the expression of PP1 緯 is up-regulated by targeting silencing PP1 偽. The expression of PP1 緯 is antagonistic and stable. At the same time, overexpression of PP1 偽 could inhibit the growth cycle of H1299 cells.
【學(xué)位授予單位】：湖南師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R341

【參考文獻】

相關(guān)期刊論文 前1條

1 王柏婧;謝秀杰;魏群;;Ⅰ型蛋白磷酸酶研究進展[J];微生物學(xué)報;2008年02期

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本文編號：1879601