【作者】 李丹丹；

【导师】 罗智；

【作者基本信息】 华中农业大学 ， 水生生物学， 2018， 硕士

【摘要】 锌是包括鱼类在内所有脊椎动物必需的微量元素,在其细胞功能和新陈代谢中起重要作用。我们最近的研究表明,锌影响黄颡鱼的脂质代谢,但进一步的机制仍然未知。相关文献指出:过量的锌诱导细胞凋亡的发生。因此,本研究的假设是锌诱导细胞凋亡且凋亡介导了锌对黄颡鱼肝脂代谢的影响。为此,本研究首先克隆得到了黄颡鱼凋亡关键基因的c DNA全长序列,分析了它们的分子特征,探讨了它们的组织表达模式;在此基础上,探讨凋亡介导锌诱导的脂代谢变化的作用和机制。主要研究结果如下:1.克隆得到了黄颡鱼Bcl2、Baxa、Baxb、Baxg、Cycs、Apaf1、Casp9、Casp3a和Casp3b基因的全长序列并分析其分子特征。Bcl2、Baxa、Baxb、Baxg、Cycs、Apaf1、Casp9、Casp3a和Casp3b基因全长依次是1098 bp、1553 bp、785 bp、1227 bp、754 bp、5028 bp、1544 bp、1831 bp和1225 bp,分别编码204、186、200、204、104、1259、442、284和285个氨基酸。与哺乳类相似,黄颡鱼Bcl2和Baxs的氨基酸序列预测由9个α螺旋组成,包含4个保守的Bcl2同源(BH)结构域(BH1、BH2、BH3、BH4)和一个碳端跨膜域。黄颡鱼Cycs蛋白序列由5个α螺旋组成,包含高度保守的细胞色素C域、几个血红素和铁原子的作用位点和三甲基赖氨酸修饰位点。黄颡鱼的Apaf1蛋白序列包含CED-3和CED-4同源域、由7个短α螺旋组成的caspase募集结构域(CARD)、Walker’s A和B盒以及WD重复域。黄颡鱼的Casp9蛋白序列包含功能前区、大小亚基、保守的天冬氨酸切割位点、CARD、Akt磷酸化域、A-X-P-X域、caspase家族组氨酸和半胱氨酸活性位点以及保守的精氨酸、亮氨酸和酪氨酸残基位点。黄颡鱼的Casp3(Casp3a和Casp3b)蛋白序列包含功能前区、大小亚基、保守的天冬氨酸切割位点、caspase家族半胱氨酸活性位点以及几个在caspase 3催化和结合过程中起重要作用的氨基酸残基。Bcl2、Baxs、Cycs、Apaf1、Casp9、Casp3a和Casp3b在黄颡鱼所有检测组织中均表达,但在不同组织中差异表达。2.探讨了凋亡介导锌诱导的脂代谢变化的作用和机制。基于电镜、TUNEL、Hoechst 33258染色和Annexin V-FITC/PI染色的观察,锌诱导了黄颡鱼肝脏和肝细胞凋亡的发生。水体锌暴露上调了p53、Cycs、Casp3a和Casp3b的基因表达。体外研究表明锌孵育上调了caspase 3活性并降低了MTP。Z-VAD-fmk和Cs A预处理缓解了锌诱导的细胞凋亡和MTP的下降。Z-VAD-fmk预处理缓解了锌暴露诱导的p53、Cycs和Casp3b基因表达的上调。而Zn和NAC对ROS没有显著影响。这些结果表明锌诱导凋亡是通过MPT下降介导的线粒体通路,不是ROS介导的线粒体凋亡通路。锌孵育显著降低了细胞内TG水平、增加了CPT I、HSL和ATGL酶活、上调了p53、cytochrome c和caspase 3b基因表达水平。锌对肝细胞的作用能被Z-VAD-fmk部分抑制住,表明锌诱导黄颡鱼肝细胞线粒体介导的凋亡通路并导致脂解。更多还原

【Abstract】 Zinc(Zn)is an essential micro-element in all the vertebrates including fish,and plays important roles in their cellular function and metabolism.Recently,our study indicated that Zn altered hepatic lipid metabolism in yellow catfish Pelteobagrus fulvidraco,but further mechanism remained unknown.Several studies pointed out that excess Zn induced the occurrence of apoptosis.Our hypothesis was that apoptosis mediated the Zn-induced changes of lipid metabolism of yellow catfish.To this end,the present study cloned and characterized the full-length c DNA sequences of several genes involved in apoptosis,and investigate the effects and potential mechanism of apoptosis mediating the Zn-induced changes of hepatic lipid metabolism.The main results were followed:1.The full-length c DNA sequences of Bcl2,Baxa,Baxb,Baxg,Cycs,Apaf1,Casp9,Casp3 a and Casp3 b involved in apoptosis were cloned and characterized in p.fulvidraco.The c DNA sequences of P.fulvidraco Bcl2,Baxa,Baxb,Baxg,Cycs,Apaf1,Casp9,Casp3 a and Casp3 b were 1098 bp,1553 bp,785 bp,1227 bp,754 bp,5028 bp,1544 bp,1831 bp and 1225 bp in length,encoding the peptides of 204,186,200,204,104,1259,442,284 and 285 amino acid residues,respectively.Similar to mammals,all Bcl2 and Baxs possessed four conserved BH domains(BH1,BH2,BH3 and BH4)and a hydrophobic domain(TM)at the C-terminus,composing of nine α-helices(α1-α9).The protein sequenses of P.fulvidraco Cycs possessed highly conserved cytochrome c domain,including several interactive sites with heme and iron ion and one modified sites of trimethyllysine,composing of five α-helices(α1-α5).The P.fulvidraco Apaf1 embraced all the characteristic features of Apaf1,including the CED-3 and CED-4 homologous regions,the seven short α-helices identified in Apaf1 CARD,the Walker’s A-and B-box consensus sequences for nucleotide binding sites,and WD repeat motifs are indicated.The predicted amino sequence of Casp9 contained a similar architecture with mammals,including a prodomain,a large subunit,a small subunit,the putative cleavage sites,caspase recruitment domain(CARD),consensus and putative Akt phosphorylation motifs,A-X-P-X motifs,caspase family histidine and cysteine active sites along with the conserved active residues ‘His’ and ‘Cys’.Furthermore,the conserved ‘Arg’ and‘Leu’ residues in prodomain and the conserved ‘Tyr’ were also found in P.fulvidraco.Casp3(Casp3a and 3b)contained a similar structure with mammals,including a prodomain,a large subunit,a small subunit,the putative cleavage sites,cysteine active sites.Several residues known to be critical in the Casp3 catalytic and binding pocket were found in P.fulvidraco.The two tryptophan residues and tyrosine residues were also found in P.fulvidraco and other vertebrates.In addition,Bcl2,Baxs,cytochrome c,Apaf1,caspase 9,caspase 3a and caspase 3b were widely expressed across the tested tissues,but at the variable levels.2.Based on the observation from TEM,TUNEL,Hoechst 33258 and Annexin V-FITC/PI staining,Zn induced occurrence of apoptosis of livers and hepatocytes in yellow catfish.Waterborne Zn exposure also increase hepatic transcriptional levels of p53,Cycs,Casp3 a and Casp3 b.The in vitro study indicated that Zn increased the caspase 3 activity and reduced the MTP in yellow catfish.Z-VAD-fmk(caspase inhibitor)and Cs A pretreatment(MTP inhibitor)attenuated the Zn-induced changes of cell apoptosis and the reduction in MTP.Z-VAD-fmk pretreatments attenuated the Zn-induced increase in transcriptional levels of p53,Cycs and Casp3 b.In contrast,Zn and NAC did not significantly influence the ROS production.These results suggested an association of Zn-induced apoptosis with not oxidative stress but MPT reduction-mediated mitochondrial apoptotic pathway.Zn incubation significantly reduced intracellular TG content,increased the activities of CPT,HSL,ATGL and up-regulated the m RNA levels of p53,cytochrome c and caspase 3b,and Zn-induced effects could partly be reversed by Z-VAD-fmk,indicating that Zn induced the mitochondrial-mediated apoptosis and resulted in hepatic lipolysis of yellow catfish.更多还原