【作者】 刘昆；

【导师】 罗海玲；

【作者基本信息】 中国农业大学 ， 动物营养与饲料科学， 2014， 博士

【摘要】 α-生育酚转移蛋白(α-tocopherol transfer protein, α-TTP)能够特异性结合并转运α-生育酚,是调控体内维生素E含量的重要因子。然而,维生素E对α-TTP表达的影响及其作用机制迄今仍未完全清楚。此外,目前α-TTP的相关研究主要集中于人类和鼠,在绵羊上尚没有相关报道。本研究以绵羊为试验对象,全面系统地分析了维生素E对绵羊α-TTP表达的影响及其可能的作用机制。试验一：克隆了绵羊α-TTP cDNA的全长基因,并对其基因序列进行了生物信息学分析。试验选用敖汉细毛羊公羊,通过cDNA末端快速扩增法克隆绵羊α-TTP cDNA全长基因,随后对其序列进行生物信息学分析。试验结果显示：绵羊α-TTP cDNA全长基因序列共1098bp,包含23bp的5’-非翻译区、226bp的3’-非翻译区以及849bp的开放阅读框,共编码282个氨基酸；绵羊α-TTP蛋白分子量约为32.01kD,具有1个Secl4p样脂质结合结构域,且蛋白不具备跨膜功能；绵羊α-TTP基因和相应的氨基酸序列和其它哺乳动物的同源性都比较高,但与非哺乳动物的同源性较低。试验二：通过在日粮中添加不同水平的维生素E,研究维生素E对绵羊α-TTP表达的影响。选取35只断奶后20-30天的滩羊公羔,随机分为五组。日粮中维生素E水平分别为0(E0)、20(E20)、100(E100)、200(E200)、2000(E2000)IU·羊-1·天-1,试验期为四个月。试验结果显示：日粮中添加高水平维生素E(E2000)可显著(P<0.05)提高血浆中维生素E的含量；同对照组相比,E200和E2000组显著(P<0.05)提高了肝脏中维生素E的含量,并可显著(P<0.05)提高滩羊肝脏中α-TTP蛋白的含量；同时,日粮中不同水平维生素E对滩羊肝脏中α-TTP基因的转录水平无显著影响。这表明日粮中添加高水平维生素E可显著提高滩羊血浆、肝脏中维生素E的含量以及肝脏中α-TTP蛋白的含量,但是对肝脏中α-TTP基因的转录水平无显著影响。试验三：通过人工合成多肽抗原结合淋巴细胞杂交瘤单克隆抗体技术制备绵羊α-TTP的单克隆抗体,并对抗体的特异性进行检测。随后,利用免疫组织化学技术对绵羊α-TTP在肝脏细胞中的定位进行观察,并检测日粮中添加不同水平维生素E后绵羊α-TTP在肝脏中的细胞学定位与分布的变化情况。试验结果表明：成功得到了绵羊α-TTP的单克隆抗体,该抗体可以特异性地结合绵羊α-TTP;绵羊α-TTP主要定位于肝脏细胞胞质,且不同水平维生素E对绵羊α-TTP的细胞学定位、分布及表达水平均无显著影响。试验四：为研究维生素E调控α-TTP基因表达的作用机制,首先对日粮中添加不同水平维生素E的绵羊肝脏组织进行基因表达谱分析,通过对差异表达基因进行GO注释和Pathway分析后筛选出绵羊肝脏中同维生素E相关的基因。另一方面,构建人α-TTP真核表达载体并在HepG2细胞中进行表达,然后利用基因表达谱芯片分析技术筛选α-TTP的相关基因。在此基础上,对维生素E及α-TTP相关基因的生物学功能进行综合分析,阐释维生素E影响α-TTP基因表达的可能作用机制。试验结果表明：共筛选出绵羊肝脏中同维生素E相关的基因探针547个;另外,同对照组相比,在HepG2细胞中表达α-TTP后引发表达水平改变的基因探针数为323个。对相关因子分析后推测：维生素E可能通过调节机体氧化水平,调控蛋白激酶C信号途径中相关基因的表达,影响转录因子活性,直接调控同其自身转运相关的因子,影响同细胞粘附、细胞炎症和细胞凋亡等相关基因的表达以及调控转位基因的表达等机制来影响α-TTP基因的表达。试验五：通过免疫共沉淀联合质谱分析技术来分离和鉴定α-TTP的互作蛋白,以研究绵羊α-TTP的互作蛋白并探讨维生素E调控α-TTP蛋白表达的可能作用机制。试验结果表明：共筛选到10个可能与α-TTP相互作用的蛋白,这些蛋白主要为同转运、能量代谢、基因表达调控和应激相关的蛋白。然而,在动物体内它们是否与α-TTP存在相互作用还有待验证。对绵羊α-TTP互作蛋白的功能进行分析后推测：维生素E可能通过配体效应、调控机体氧化应激水平以及改变转位因子构象等机制来影响α-TTP蛋白的表达。综上所述,本论文全面系统地研究了维生素E对绵羊α-TTP表达的影响及其作用机制,这些结果将有助于对绵羊α-TTP功能及维生素E转运调控机制的理解。更多还原

【Abstract】 α-tocopherol transfer protein (α-TTP) is a protein that exhibits a marked ligand specificity and selectively recognizes of α-tocopherol and plays a key role in regulating concentrations of vitamin E. Despite the central roles that α-TTP plays in maintaining vitamin E adequacy, the available information is insufficient for a complete understanding of the mechanisms that how α-TTP is regulated by vitamin E supplementation. The studies of α-TTP have been mainly focused on human, mice and rats, however, little is known about ovine α-TTP. In the present study, α-TTP was first investigated in sheep and especially focused on the regulation of ovine α-TTP by dietary vitamin E supplementation and its possible mechanisms.Experiment1:Molecular cloning and analysis of the full-length cDNA of ovine α-TTP gene. The full-length cDNA of ovine p a-TTP gene was cloned from the liver tissue of Aohan fine-wool sheep by using of rapid amplification of complementary DNA ends. Bioinformatic analysis was then applied to analyze the gene and protein sequences of ovine α-TTP. As a result, the ovine α-TTP gene was1098bp in nucleotide which contained23bp5’-untranslated region (UTR),226bp3’-UTR and849bp open reading frame (ORF) that encoded a basic protein of282amino acids. Further bioinformatic analysis indicated that the ovine α-TTP was calculated to have a molecular mass of32.01kDa and it had a Secl4p-like lipid-binding domain. Besides, the ovine α-TTP was not a transmembrane protein. According to its gene and protein sequences, the ovine α-TTP had a high homologous of both nucleotide and amino acid sequences with the corresponding α-TTP gene from other mammals, however, the homologies were relatively low between sheep and other non-mammals.Experiment2:Effect of dietary vitamin E supplementation on the expression levels of ovine α-TTP. Thirty-five local male lambs of Tan sheep,20to30days after weaning, were randomly divided into five groups (n=7). The sheep were fed with diets supplemented with0(E0),20(E20),100(E100),200(E200) and2000(E2000) IU sheep-1d-1vitamin E for120days. The result showed that sheep fed with a high vitamin E diet (E2000) had significantly (P<0.05) higher plasma vitamin E concentration. Similarly, E200and E2000groups had significantly (P<0.05) higher hepatic vitamin E and α-TTP protein level compared to the control group (E0), however, no significant change was observed in ovine α-TTP gene expression among groups. Overall, the result indicated that high levels of vitamin E supplementation significantly (P<0.05) increased the concentrations of vitamin E in the plasma and liver as well as ovine liver α-TTP protein level, however, dietary vitamin E supplementation did not affect ovine α-TTP gene expression.Experiment3:Preparation of monoclonal antibody for ovine α-TTP. The amino acid sequenc of ovine α-TTP was first analyzed and two peptide immunogens with high specificity were selected and artificially synthesized. Then sheep α-TTP monoclonal antibody was produced by using lymphocyte hybridoma monoclonal antibody technology. In the end, the produced monoclonal antibody was purified and confirmed by different assays. Besides, immunohistochemical studies were also applied to analyze the distribution of a-TTP in ovine liver and the ovine a-TTP expression levels under different vitamin E supplementation. The result showed that a specific monoclonal antibody was successfully obtained which was proved to be effective in recognizing ovine a-TTP. Immunochemical staining also showed that a-TTP staining was mainly cytosolic and the result of immunohistochemistry revealed that no significant effect of vitamin E on ovine a-TTP distribution and expression levels in liver cells were observed.Experiment4:In order to study the mechanism of how ovine a-TTP gene expression was regulated by vitamin E, microarray technology was applied to screen differential expression genes in ovine liver among groups with different dietary vitamin E supplementation. On the other hand, the a-TTP vector was expressed in HepG2cell and a-TTP related genes were also screened by using microarray technology. Based on this, the function and pathway of both vitamin E and a-TTP related genes were analyzed in a comprehensive way to clarify how ovine a-TTP gene expression was regulated by vitamin E. Results suggested that547gene probes which might interact with vitamin E in ovine liver were obtained. Besides,323gene probes were identified to be regulated after a-TTP was expressed in HepG2cell. Based on the analysis of these genes, it was speculated that vitamin E might affect a-TTP gene expression by modulating the oxidation level or affecting genes implicated in protein kinase C signaling, or regulating the expression of transcription factors. Besides, vitamin E might also regulate a-TTP gene expression by regulating genes that related to the uptake and degradation of tocopherols, or genes which connected to adhesion, inflammation and apoptosis as well as some translocation-related genes.Experiment5:In order to elucidate how ovine a-TTP protein expression was regulated by vitamin E, ovine a-TTP interacting protein was screened using the method of co-immunoprecipitation coupled with mass spectrometry.10proteins that might interact with ovine a-TTP were identificated and these proteins were mainly involved in transportation, energy metabolism, gene expression regulation and stress reaction. However, whether they interact with ovine a-TTP in vivo or not still needs to be verified. It was possible that vitamin E might regulate a-TTP protein expression by acting as a ligand or by modulating the oxidative stress level. It was also very likely that vitamin E could affect a-TTP protein expression via changing the conformation of some translocation-related factors.Collectively, the present study investigated the effect of dietary vitamin E supplementation on ovine a-TTP expression for the first time and analyzed its mechanism in a comprehensive way. The findings will greatly enhance our understanding of the biological functions of ovine a-TTP and regulation mechanism of vitamin E transportation.更多还原