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目的:多倍性是物种形成的重要机制,决定一些重要器官细胞产生的数量和功能,而且与某些病理过程(如恶性肿瘤)的发生有密切关系。我们通过建立相对同步化的多倍体细胞模型,已经证实mTOR/S6K1参与多倍体细胞周期的调控。本课题主要研究mTOR下游的另一个重要信号分子4E-BP1是否也参与细胞的倍体化调控。方法:诺考达唑诱导Dami细胞建立相对同步化的多倍体细胞模型,Western-blot分析多倍体细胞模型中mTOR/4E-BP1通路信号分子表达和磷酸化修饰位点的变化,流式细胞仪双荧光分析4E-BP1不同结构域磷酸化位点修饰与细胞周期各时相的关系。结果:诺考达唑诱导的Dami细胞可作为相对同步化的多倍体细胞周期模型,在二倍体和多倍体细胞周期中,mTOR表达增加及第2448位丝氨酸位点磷酸化发生在G1期进入S期,4E-BP1的第37,46位苏氨酸和第65位丝氨酸位点磷酸化发生在G2/M期。结论:mTOR/4E-BP1通路参与多倍体细胞周期的调控。
Objective: Polyploidy is an important mechanism of species formation, which determines the quantity and function of cells in some important organs and is closely related to the occurrence of certain pathological processes (such as malignant tumors). By establishing a relatively synchronized polyploid cell model, we have shown that mTOR / S6K1 is involved in the regulation of the polyploid cell cycle. This topic mainly studies whether mTOR downstream of another important signal molecule 4E-BP1 is also involved in cell ploidy regulation. METHODS: Nocodazole induced Dami cells to establish a relatively synchronized polyploid cell model. Western blot analysis of mTOR / 4E-BP1 pathway signaling molecules and phosphorylation sites in the polyploid cell model was performed by flow cytometry Dual - Fluorescence Analysis of Cytosolic Phosphorylation Sites in Different Regions of 4E-BP1 during Cell Cycle. RESULTS: Dacron-induced Dami cells served as a relative synchronized polyploid cell cycle model with increased mTOR expression at the diploid and polyploid cell cycle and phosphorylation of serine at position 2448 at G1 Into the S phase, 4E-BP1 37,46th threonine and the 65th serine site phosphorylation occurred in the G2 / M phase. Conclusion: mTOR / 4E-BP1 pathway is involved in the regulation of polyploid cell cycle.