SP600125诱导鱼类细胞多倍化的机制及应用研究

Polyploidization by artificial induction plays an important role in germplasm innovation and breeding of new varieties. In our previous research, SP600125 has been used to induce polyploidization of fish cells in vitro, and a stable fish tetraploid cell line has been obtained. However, it is still unclear about the mechanism of SP600125-induced polyploidization. By transcriptome analysis, we have revealed that SP600125 causes modulation of cell cycle checkpoints, which might be the key reason of fish cell polyploidy induction. Based on the existing results, this project is intended to further investigate: (1) What is the chromosome behavior of cell in polyploidization induced by SP600125 in fish; (2) What are the regulation effects of SP600125 on the centromere protein and relevant genes; (3) How to establish the behavior of p53 gene in stimulating and influencing the cellular fate under the induction of SP600125; (4) Owing to the promising developmental potential of SP600125-induced tetraploid cells, another focus of this project is to explore the techniques and its implementation plan being associated with SP600125 as a chemical inducer, which will be conducted by directly inducing the fertilized eggs or gonad in fish. The results of this project are useful to master the mechanism of SP600125-induced polyploidization, and provide theoretical cornerstones and new practicable techniques for the artificial induction polyploid breeding of fish in a more efficient way.

人工诱导多倍体在种质创新和新品种选育上发挥了重要作用。前期研究发现，小分子化合物SP600125能有效诱导鱼类细胞发生多倍化，并成功获得了SP600125诱导的同源四倍体鲫细胞系。然而，SP600125诱导鱼类细胞多倍化的机制尚不清楚。转录组分析发现，SP600125对细胞周期检查点具有多重调控，并且可能是其实现鱼类细胞多倍化诱导的关键分子机制。在此基础上，本项目拟进一步深入研究：(1)SP600125诱导多倍化过程中细胞的染色体行为；(2)SP600125诱导过程中纺锤体检验点蛋白及其信号通路调控；(3)SP600125诱导过程中p53对细胞多倍化的调控；(4)SP600125作为化学诱导剂制备多倍体鱼的技术途径。本项目旨在挖掘SP600125诱导多倍化相关的基因和调控的改变，较为深入地了解SP600125诱导鱼类多倍化的潜能，为鱼类人工诱导多倍体培育提供新的策略和方法。

前期研究发现小分子化合物SP600125能有效诱导鱼类细胞发生多倍化，并成功获得SP600125诱导的同源四倍体鲫细胞系。然而，SP600125诱导鱼类细胞多倍化的机制尚不清楚。本项目结合荧光示踪观察，证实SP600125在细胞增殖过程中直接影响染色体行为并导致有丝分裂M期阻滞。在此基础上，通过RNA-seq全面挖掘SP600125诱导细胞多倍化和细胞稳态维持相关的信号调控网络和关键功能基因。结合蛋白抑制剂孵育及过表达检测等对基因的功能分析，发现SP600125诱导增强了p53信号通路的M期阻滞，并导致SAC检测点信号削弱引发的有丝分裂滑移，证实SP600125通过对细胞周期检验点多重调控从而实现鱼类细胞多倍化。解析了SP600125诱导多倍化进程中的细胞适应性，发现急性免疫应激参与SP600125胁迫下的细胞内稳态维持调节。发现SP600125诱导的四倍体细胞与二倍体、四倍体鱼细胞在能量代谢及调节上差异显著，而二倍体和四倍体之间则表现出较为稳定的细胞周期调控基因的共同表达趋势，并筛选出适于鱼类多倍体荧光定量分析的内参基因。发现SP600125作为化学诱导剂对鱼骨骼发育存在致畸风险，同时利用SP600125对细胞分裂的阻滞作用建立了一种制备染色体标本的新方法。相关研究成果为SP600125在鱼类倍性操作和肿瘤研究中的应用提供了重要理论参考。

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