p53介导游离棉酚致鲤肝脏中毒的分子机制研究

The p53 is such a transcription factor, that plays an important role in translating stress signals into classic processes such as apoptosis, cell cycle arrest, DNA repair and senescence. Free gossypol has the potential to inbibit cell proliferation in various types of cancers through p53 regulation. In Cyprinus carpio, free gossypol can induces apoptosis and disorder of cell cycle in hepatocytes. However, the role of p53 is still not fully understood in hepatotoxicity induced by free gossypol in C. carpio. In previous study, we have obtained the partial cDNA sequences of p53 and its target genes of the C. carpio. Here, in vivo and in vitro, we will firstly clone and sequence p53 and its target genes, and examine the role of p53 in the regulation of the cell cycle in C. carpio, by analyzing how p53 knockdown and p53 inhibitor affects the expression of its target genes. Secondly, we will analyze the concentration of free gossypol in hepatocyte, and clarify the correlation between the toxicokinetic process of free gossypol and hepatic impairment. Thirdly, we will elucidate the regulatory mechanism of p53 base on the gene mutation, mRNA transcription, protein levels and posttranslational modification by DNA sequencing, Real-time quantitative PCR and Western blot. This study can elucidate the regulatory mechanism of p53 undergo free gossypol in C. carpio, but also can provide an effective method for evaluating the security of cottonseed meal in aquatic feed and detoxication therapy for free gossypol in fish.

p53是在细胞凋亡和细胞周期中发挥重要调控作用的一种转录因子。在p53介导下，游离棉酚(free gossypol，FG)可诱导人类癌细胞凋亡，引起癌细胞周期阻滞。在鲤中，FG不仅可诱导鲤肝细胞发生凋亡，也可引起肝细胞周期异常。但在此过程中，p53所发挥的调控功能尚不清楚。本项目在已获得鲤p53及其下游靶基因部分序列基础上，从在体和离体水平，综合采用RNAi、Real-time PCR、Western-blot、DNA直接测序法、高效液相色谱检测和流式细胞分析等技术，明晰p53对鲤肝细胞凋亡和细胞周期的调控功能；揭示FG毒理动力学特征与肝细胞中毒的关系；从基因突变、mRNA转录、蛋白翻译和蛋白修饰水平阐明p53对FG的应答机理，并解析p53与下游靶基因在FG致肝中毒过程中表达模式的相关性。预期研究结果可从分子水平揭示FG对鲤的致毒机制，为研发鱼类FG解毒新技术提供理论依据。

p53是在细胞凋亡和细胞周期中发挥重要调控作用的一种转录因子。在p53介导下，游离棉酚可诱导人类癌细胞凋亡，引起癌细胞周期阻滞。棉粕作为水产饲料中重要蛋白来源之一，但其含有的游离棉酚对鱼类毒性机制尚不完全清楚。本研究以鲤为研究对象，研究了p53在游离棉酚对鲤致毒中的作用机制。研究发现，游离棉酚在鲤体内“首过效应”较强，主要蓄积在肝脏和肾脏，血液中游离棉酚含量较低。鲤单次灌喂醋酸棉酚，短时间内引起了肝脏生理性变化，未引起肝脏病理性损伤以及肝细胞凋亡。不同剂量连续灌喂鲤游离棉酚，血清转氨酶活性升高，肝脏肿大，肝细胞核直径显著增大，肝细胞出现凋亡，肝脏脂肪含量增加；游离棉酚诱导鲤p53高表达，通过激活下游Bax-caspase9-caspase3/caspase7和fas-caspase8-caspase3/caspase7两条通路发生肝细胞凋亡。饲料中添加不同剂量游离棉酚投喂鲤8周后，鲤生长性能未受到影响，肝脏脂肪含量增加，肝脏一定程度受损，但未引起肝细胞凋亡。研究结果显示，鲤对游离棉酚具有一定耐受性，游离棉酚对鲤肝脏致毒呈现浓度与时间依赖性，通过p53介导作用引起肝细胞凋亡，其凋亡过程可能受到营养与饥饿影响，在饥饿与投饵条件下游离棉酚均可引起鲤肝脏脂肪蓄积。本研究对进一步理解游离棉酚对鱼类致毒机制，通过营养调控技术减弱游离棉酚毒性具有重要参考价值。