蛋白磷酸酶PP1α和PP1γ在H1299细胞中的表达水平和抑瘤功能分析

发布时间：2018-05-12 18:05

本文选题：PP1α + PP1γ　；参考：《湖南师范大学》2011年硕士论文

【摘要】：蛋白质的磷酸化与去磷酸化过程是生物体内一种普遍存在的调节方式,几乎涉及所有的生理及病理过程,并且在细胞信号的传递过程中占有极其重要的位置。蛋白质的磷酸化和去磷酸化反应分别由蛋白激酶和蛋白磷酸酶催化。蛋白激酶由一个庞大的并且功能极其多样的基因家族所构成,相比之下,蛋白磷酸酶则有着较少的数量和种类。蛋白磷酸酶1(PP1)的催化亚基能与大于150种的调节亚基形成复合物,这种复合物的构造使得PP1c转变成很多不同的形态,从而具有底物特异性、独特的亚细胞定位和特异性的调节功能。这使得大量的依靠PPl的细胞功能通过独立的机制得到控制。我们以H1299为实验材料,通过运用脂质体转染的方法,建立了H1299-pCI-neo、H1299-pCI-PP1α、H1299-pLKO.1和H1299-pLKO.1-PP1α等稳定表达细胞株,通过Western Blot的方法鉴定和检测了PP1α和PP1γ的相对表达情况,我们发现：当过表达PP1α时,H1299细胞中的PP1γ的表达量出现了明显的下调,而当靶向沉默PP1α时,H1299细胞中的PP1γ表达水平则出现了明显的上调,并且,PP1β和PP1γ的表达似乎维持着总量的平衡,二者呈现出拮抗表达的关系。通过RT-PCR,我们验证了在mRNA水平也同样存在着这种关系,这说明PP1α和PP1γ拮抗表达的关系在mRNA水平就受到了严格地调控。我们还通过细胞增殖实验和裸鼠荷瘤实验,对H1299、H1299-pCI-neo、H1299-pCI-PP1α三种稳定细胞株进行了鉴定,发现过表达PP1α能明显的延缓细胞周期并抑制肿瘤细胞的生长,同时,我们也通过Western Blot检测了几种基本的周期蛋白的表达,发现CyclinA/B/E与PP1α的表达具有协同效应,而其具体的作用机制有待进一步研究验证。综上,我们可以得出以下结论：在H1299细胞中,过表达PP1α后PP1γ的表达明显下调,相反靶向沉默PP1α则PP1γ的表达明显上调,二者的表达具有拮抗关系,并维持着总量的稳定。同时过表达PP1α能够阻抑H1299细胞的生长周期的进行,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 伪, H1299-pLKO.1 and H1299-pLKO.1-PP1 伪 were established by using liposome transfection. The relative expression of PP1 伪 and PP1 纬 was identified and detected by Western Blot. We found that the expression of PP1 纬 in H1299 cells was significantly down-regulated when PP1 伪 was over-expressed, while the expression level of PP1 纬 in H1299 cells was up-regulated when PP1 伪 was silenced, and the expression of PP1 尾 and PP1 纬 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 伪 and PP1 纬 is strictly regulated at the mRNA level. We also identified three stable cell lines H1299-pCI-neoH1299-pCI-PP1 伪 by cell proliferation test and tumor-bearing assay in nude mice. It was found that overexpression of PP1 伪 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 伪 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 纬 is down-regulated after overexpression of PP1 伪, whereas the expression of PP1 纬 is up-regulated by targeting silencing PP1 伪. The expression of PP1 纬 is antagonistic and stable. At the same time, overexpression of PP1 伪 could inhibit the growth cycle of H1299 cells.
【学位授予单位】：湖南师范大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R341

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