【作者】 曹艳姿；

【导师】 吉红；

【作者基本信息】 西北农林科技大学 ， 水生生物学， 2010， 硕士

【摘要】 高不饱和脂肪酸能够维持鱼类细胞膜结构和机能的完整性、保持某些酶活性、构成前列腺素前体及增强免疫系统功能。有学者研究了不同脂肪酸及脂肪源饲料对淡水鱼生长、脂肪酸组成及体成分的影响,发现HUFA或鱼油具有促进生长及降低体脂含量的作用。其影响体脂沉积的作用是由于HUFA抑制了体脂合成还是转运,或者是促进了脂质分解尚不清楚,相关机理的研究需要深入开展。研究脂代谢相关基因:LXRα、SREBP-1、FAS、ACC、LPL mRNA表达变化,有助于深入理解鱼类脂质代谢的分子机理。在进行脂肪组织在体发育规律研究的同时,对前体脂肪细胞进行离体培养,监测体外培养过程中增殖分化的规律,探讨脂肪组织发生与增生的规律和机制,是研究动物体内脂肪沉积规律的必要手段及有效途径。本试验以草鱼为研究对象,从脂代谢相关基因的克隆入手,结合前体脂肪细胞离体培养及HUFA在体饲喂,系统的探讨了HUFA对草鱼脂肪细胞增殖分化的发育及肝胰脏脂质代谢的影响作用。得出如下结果:1.成功克隆草鱼脂质代谢基因LXRαcDNA(GenBank注册号为FJ965309)序列及SREBP-1(GenBank注册号为GU339498)、FAS(GenBank注册号为GQ466046)、ACC(GenBank注册号为HM142590)基因的部分cDNA序列,大小分别为1239bp、899bp、709 bp、467 bp。其中LXRα基因与其它物种同源性为70.7%~100%,与鲤鱼和斑马鱼的同源性高达100%和94.6%;LXRα基因在草鱼肝胰脏、肌肉、心脏、鳃、精巢、肾脏、脑、脾脏、肠、腹腔脂肪等组织中广泛表达,且存在组织差异,在精巢中表达丰度最高,在肌肉中表达丰度最低。2.草鱼前体脂肪细胞可自发充脂;经诱导培养后,脂肪细胞充脂率提高,特异性标志基因表达增强。3.DHA促进草鱼前体脂肪细胞增殖,抑制其分化。4.饲料中HUFA显著影响鱼体脂肪酸组成,HUFA显著提高草鱼腹腔脂肪组织中C22:6n-3(p<0.01)、HUFA(p<0.05)含量;显著升高肌肉组织中n-3(p<0.01)、HUFA(p<0.05)含量,而降低C20:4n-6含量(p<0.05);除脑组织外,肝胰脏、肌肉、腹腔脂肪组织中n-3/ n-6均显著升高(p<0.05)。与对照组相比,HUFA显著降低腹腔脂肪指数、肝胰脏脂质含量,显著升高肝胰脏T-AOC活性(p<0.05),肝胰脏LPL活性显著降低(p<0.05)。同时,HUFA还显著抑制了草鱼肝胰脏LXRα、SREBP-1c、FAS、ACC、LPL mRNA的表达。通过以上研究表明,饲料HUFA对草鱼脂质代谢具有明显的调控作用,通过抑制生脂基因表达、外源脂质进入肝胰脏及脂肪细胞分化,降低肝脂和腹脂含量,同时显著影响组织脂肪酸组成,改善肉质品质。更多还原

【Abstract】 Highly unsaturated fatty acid can maintain the cell membrane structure of fish, the function integrity, certain enzyme activities, the constitution prostaglandin precursor and the enhancement immune system function. Some researchers have studied the different fatty acid and the fat source dietary influence the growth performance, fatty acid composition and body composition of fresh water fish, discovered that HUFA or fish oil have the function that promoting the growth performance and reducing body fat content. But its influence body fat deposition’s function is because HUFA inhibited the lipid metabolism or transport, or promoted lipidolysis was not still clear, the further evaluation of the related mechanism was reqiured in freshwater fishes.We study the change on mRNA expresson of LXRα、SREBP-1、FAS、ACC、LPL which related to lipid metabolism in feeding process, its helpful to understand the molecular mechanism of fish lipid metabolism. The essential method about reseach of the rule of animal fat deposition was adipocytes culturing in vitro and monitoring the process of adipocyte proliferation with reseaching about development regulation of adipose tissue.Grass carp was used as the experimental animal in this study. After being treated with HUFA directly, the pattern of the change of gene expression which related lipid metabolism and cultured adipocytes in vitro was investigated. Based on the study on the rules of the adipocytes proliferation and differentiation, it is aimed to work over the effect and mechanism of HUFA on adipocyte development in grass carp, which is important to elucidate the development mechanism of adipocyte tissue, and beneficial to provide new train of thought and theoretic foundation on the regulation of lipid storage in cultured fish.The main results were as follows:1. The cDNA sequence of Liver X Receptorα(LXRα)(GenBank accession number FJ965309 ), and the partial cDNA sequence of sterol regulatory element- binding protein 1 (SREBP-1) (GenBank accession number GU339498 ), fatty acid sythetase(FAS)(GenBank accession number GQ466046 ), acety1 COA carboxylase (ACC) (GenBank accession number HM142590) were cloned.The obtained sequence is 1230bp, 899bp, 709bp, 467bp in length separately. Homology is 70.7%~100% with other species. Widely expressed in hepatopancreas, muscle, heart, gill, spleen, intestin, brain, kidney, testis, and intrapaneal fat body (IPF),with the highest expression level of LXRαgene was found in testis, lowest one in muscle (p<0.05).2. Preadipocytes active and differentiate spontaneously. After inducting the preadipocytes filled with lipid-rich droplets at a higher ratio, and the adipogenic gene expression was enhanced.3. DHA promote preadipocytes of grass carp proliferation, inhibit its differentiation.4. Dietary HUFA were very effectively incorporated into the fish body. Analysis of fatty acid indicated that a higher content of C22:6n-3(p<0.01) and HUFA (p<0.05) were found in intraperiotenal fat (IPF), and a higher content of n-3 (p<0.01) and HUFA (p<0.05) but lower C20:4n-6 content (p<0.05) in muscle of HUFA group. The content of n-3/n-6 was significantly higher in the hepatopancreas, IPF, and muscle in fish fed the HUFA diet compared to the control diet. The content of IPF and hepatopancreas lipid (p<0.05) was lower in the HUFA group. On the other hand, a significantly higher T-AOC activity, the lower LPL activity and LPL,LXRα, SREBP-1c, FAS, ACC mRNA expression(p<0.05) was observed in the hepatopancreas of fish fed the HUFA diet.From above, our study suggest that HUFA significantly regulate lipid metabolism of grass carp, through suppresses the expression of lipogenic gene mRNA,the exogenous application lipid enter to hepatopancreas and adipocytes differentiation, reduces the liver fat and abdomen fat content, fatty acid composition is affected by fatty acid composition of the diet, improving the quality level of the fish.更多还原