羅非魚(yú)PPARγ在脂代謝中的調(diào)節(jié)作用及其轉(zhuǎn)錄活性
發(fā)布時(shí)間：2018-08-09 12:54
【摘要】：尼羅羅非魚(yú)(Oreochromis niloticus)生長(zhǎng)性能好且抗逆性強(qiáng),已成為世界性養(yǎng)殖品種。作為一種重要的模式物種,其基因組測(cè)序已經(jīng)完成,有利于以其為模型開(kāi)展魚(yú)類營(yíng)養(yǎng)生理研究。過(guò)氧化物體增殖物激活受體γ(Peroxisome proliferator activated receptor gamma, PPARy)是動(dòng)物脂類代謝關(guān)鍵調(diào)節(jié)器,它廣泛存在于脊椎動(dòng)物中。然而,目前尚缺乏有關(guān)魚(yú)類PPARy生理功能、分子結(jié)構(gòu)及轉(zhuǎn)錄活性等方面的研究。本研究以尼羅羅非魚(yú)為模型,采用分子克隆、細(xì)胞培養(yǎng)、熒光定量PCR、組織學(xué)分析、蛋白印跡、轉(zhuǎn)錄組學(xué)、雙熒光素報(bào)道系統(tǒng)、啟動(dòng)子捕獲和代謝物含量測(cè)定等手段針對(duì)PPARy進(jìn)行以下四方面的研究：1)羅非魚(yú)PPARy在調(diào)節(jié)脂質(zhì)內(nèi)穩(wěn)態(tài)中的作用；2)羅非魚(yú)PPARy結(jié)構(gòu)分析及轉(zhuǎn)錄活性研究；3)哺乳動(dòng)物PPARy激活劑Rosiglitazone和抑制劑GW9662對(duì)羅非魚(yú)脂代謝的影響,和4)PPARy靶基因ACOX1的克隆及其營(yíng)養(yǎng)調(diào)控。論文的主要結(jié)果與結(jié)論如下1.尼羅羅非魚(yú)PPARy在脂質(zhì)內(nèi)穩(wěn)態(tài)中的調(diào)節(jié)作用自然選擇不僅賦予了動(dòng)物在脂類物質(zhì)豐富時(shí)貯存脂類的能力,同時(shí)也賦予了其在外界脂類攝入不足時(shí)進(jìn)行體內(nèi)脂類合成的能力。然而魚(yú)類是如何利用自身的脂代謝系統(tǒng)來(lái)應(yīng)對(duì)低脂食物和高脂食物的不同營(yíng)養(yǎng)條件仍未得到很好的闡明。本研究通過(guò)高脂飼料和低脂飼料投喂法來(lái)探究尼羅羅非魚(yú)在脂類攝入過(guò)量和不足這兩種情況下是如何維持脂類內(nèi)穩(wěn)態(tài)的,以及PPARy在這一過(guò)程中扮演了何種角色。實(shí)驗(yàn)結(jié)果顯示,持續(xù)10周投喂脂肪含量分別為1%,7%和13%三種飼料后,三組之間的生長(zhǎng)率(Growth rate),肝體比(Hepatic somatic index),血清、肝臟、肌肉和脂肪組織的甘油三酯含量均無(wú)顯著差異,但是體脂肪含量和脂體比隨著飼料中脂肪水平的增加而增加。熒光定量PCR、轉(zhuǎn)錄組學(xué)、蛋白免疫印跡等實(shí)驗(yàn)結(jié)果表明,肝臟是應(yīng)對(duì)低脂投喂的主要器官,主要表現(xiàn)為糖酵解速率增加和脂肪酸從頭合成能力增強(qiáng)；在高脂條件下,脂肪組織通過(guò)吸收更多游離脂肪酸并增加脂肪水解,進(jìn)而激活PPARγ,被激活的PPARγ進(jìn)一步促進(jìn)脂肪細(xì)胞增殖,從而使脂肪細(xì)胞能在不變大的情況下貯存更多甘油三酯,此研究結(jié)果表明羅非魚(yú)PPARy有著與哺乳動(dòng)物相似的功能。此研究首次較為系統(tǒng)的闡明了魚(yú)類應(yīng)對(duì)脂肪攝入不足和過(guò)量時(shí)的生理調(diào)控機(jī)制,豐富了我們對(duì)魚(yú)類生理學(xué)的認(rèn)識(shí)。2.尼羅羅非魚(yú)PPARy結(jié)構(gòu)分析及轉(zhuǎn)錄活性研究過(guò)氧化物酶體增殖物激活受體y (Peroxisome proliferator activated receptor gamma, PPARy)是動(dòng)物脂類代謝的關(guān)鍵調(diào)節(jié)器,前節(jié)表明尼羅羅非魚(yú)PPARy參與調(diào)節(jié)羅非魚(yú)脂肪細(xì)胞增殖,分子結(jié)構(gòu)決定了其生理功能,因此在此基礎(chǔ)上,我們進(jìn)一步對(duì)PPARy的分子結(jié)構(gòu)和轉(zhuǎn)錄活性進(jìn)行了分析。本研究首先獲得了尼羅羅非魚(yú)PPARy全長(zhǎng)(NtPPARy),并通過(guò)構(gòu)建NtPPARy和NtRXRa表達(dá)載體,及FABP4啟動(dòng)子驅(qū)動(dòng)的報(bào)道載體在HEK-293細(xì)胞系中開(kāi)展了轉(zhuǎn)錄活性研究。結(jié)果發(fā)現(xiàn)：NtPPARy存在兩種轉(zhuǎn)錄本,二者在5’-非編碼區(qū)存在差異,與哺乳動(dòng)物比較后發(fā)現(xiàn),NtPPARy的LBD (Ligand binding domain)多了39個(gè)氨基酸殘基,使得羅非魚(yú)LBD區(qū)比哺乳動(dòng)物多3個(gè)alpha螺旋結(jié)構(gòu)。組織表達(dá)模式結(jié)果發(fā)現(xiàn)兩種轉(zhuǎn)錄本在11種組織中呈現(xiàn)出不同的分布模式,但兩者均在肝臟、腸和腎臟中高表達(dá)。轉(zhuǎn)錄活性研究結(jié)果顯示NtPPARy與NtRXRa一起參與尼羅羅非魚(yú)FABP4基因的轉(zhuǎn)錄調(diào)節(jié),與人類中的轉(zhuǎn)錄調(diào)節(jié)方式相似�？傊�,PPARy的DBD (DNA binding domain)高度保守,但LBD保守性相對(duì)較弱,而且羅非魚(yú)PPARy與RXRa一起參與諸如FABP4等靶基因的轉(zhuǎn)錄調(diào)節(jié)。在獲得尼羅羅非魚(yú)細(xì)胞系的基礎(chǔ)上,本研究所構(gòu)建的質(zhì)粒系統(tǒng)將會(huì)是未來(lái)研究魚(yú)類PPARy功能的有效工具。3.哺乳動(dòng)物PPARy激活劑Rosiglitazone和抑制劑GW9662對(duì)羅非魚(yú)脂代謝的影響功能獲得(Gain of function)和功能缺失(Loss of function)是研究基因功能的有效策略,利用激活劑和抑制劑可以快速實(shí)現(xiàn)功能獲得和功能缺失,因此我們嘗試通過(guò)哺乳動(dòng)物PPARy激活劑Rosiglitazone (Rosi)和抑制劑GW9662(GW9)來(lái)激活和抑制羅非魚(yú)PPARy,并在此基礎(chǔ)上探討羅非魚(yú)PPARy的生理功能。本研究首先設(shè)計(jì)二種脂肪水平的飼料,即4%(Standard diet, SD)和15%(High fat diet,HFD),每種水平下包括對(duì)照組,Rosi (15mg/kg)組,和GW9 (10mg/kg)組,共6組飼料,養(yǎng)殖6周后采樣分析。結(jié)果發(fā)現(xiàn)HFD組的脂體比顯著高于SD組,肝體比和肝臟中TG含量也高于SD組,表明高脂導(dǎo)致肝臟脂肪沉積過(guò)量；HFD組血清TG含量也顯著高于SD組,表明脂質(zhì)內(nèi)穩(wěn)態(tài)失衡,成功獲得我們所需的脂肪肝模型。但無(wú)論是在低脂還是高脂背景下,Rosi和GW9的添加對(duì)羅非魚(yú)生長(zhǎng)和代謝指標(biāo)均無(wú)顯著影響。因此我們采用腹腔注射這一更為直接的給藥方式進(jìn)行二次實(shí)驗(yàn),Rosiglitazone注射劑量為：0(DMSO)、0.6mg/kg、18mg/kg和54 mg/kg;GW9662注射劑量為：0(DMSO)、0.4mg/kg、12mg/kg和36 mg/kg,連續(xù)注射三天后采樣分析。腹腔注射GW9662對(duì)血清中甘油三酯(Triglyceride, TG)、游離脂肪酸(Free fatty acid, FFA)和葡萄糖(Glucose)均無(wú)顯著影響。注射Rosiglitazone對(duì)血糖無(wú)影響,但是顯著下調(diào)了TG含量,上調(diào)了FFA含量。熒光定量PCR分析發(fā)現(xiàn)肝臟中脂肪酸合成,TG合成和VLDL關(guān)鍵蛋白基因表達(dá)量均下降,表明TG含量下降主要?dú)w功于肝臟脂質(zhì)合成和分泌減少,FFA的升高則歸功于肌肉中脂肪水解增強(qiáng)。但是Rosi沒(méi)有影響PPARγ在脂肪組織、肝臟和肌肉中的表達(dá),是否調(diào)節(jié)了PPARγ的轉(zhuǎn)錄后修飾,并影響了其蛋白活性從而引起相應(yīng)的生理效應(yīng)則需進(jìn)一步研究。以上結(jié)果表明,短期Rosi注射能夠促進(jìn)羅非魚(yú)肌肉脂肪水解。4. PPARγ靶基因ACOX1的克隆及其營(yíng)養(yǎng)調(diào)控過(guò)氧化物酶體是合成多種脂類信號(hào)分子的場(chǎng)所。過(guò)氧化物酶體中的代謝活動(dòng)受到PPARγ的調(diào)節(jié),酯酰輔酶A氧化酶1(Acayl-CoA Oxidase 1, ACOX1)是過(guò)氧化物酶體β-氧化的第一限速酶,它參與酯酰輔酶A至2-順式烯酰輔酶A的催化過(guò)程,而過(guò)氧化物酶體β-氧化過(guò)程為信號(hào)分子的合成提供底物。ACOX1是PPARγ的靶基因之一,因此闡明ACOX1的結(jié)構(gòu)和功能對(duì)研究PPARγ如何調(diào)控過(guò)氧化物體信號(hào)分子的合成具有重要作用。本研究首次在尼羅羅非魚(yú)中克隆獲得了ACOX1基因,發(fā)現(xiàn)其具有兩種轉(zhuǎn)錄本,分別命名為ACOXli1和ACOXli2,它們編碼的蛋白均由661個(gè)氨基酸殘基組成。兩亞型的編碼區(qū)由14個(gè)外顯子組成。ACOXli1的89-193號(hào)位氨基酸由外顯子E3b編碼,而ACOXli2,的貝由E3a編碼,同源比對(duì)分析發(fā)現(xiàn)ACOXli1的保守性明顯高于ACOXli2,提示ACOXli1的功能可能比ACOXli2的功能更為重要。二者的mRNA組織分布模式表明：ACOXi1在肝中的表達(dá)量最高,其次為腎臟和腦；而ACOXli2在腎臟中表達(dá)量最高,其次為肝臟,兩種亞型在白肌、鰓、腦和腎臟中的表達(dá)量呈顯著差異,在白肌和腎臟中ACOXli2顯著高于ACOXlil,而在腦和鰓中則相反；在饑餓36小時(shí)、飽食后1、3、8、24小時(shí)的營(yíng)養(yǎng)狀態(tài)研究中發(fā)現(xiàn)：兩種亞型在肝中對(duì)營(yíng)養(yǎng)狀態(tài)做出了相同的反應(yīng)模式,腎臟ACOXlil與肝中的反應(yīng)模式趨同,而ACOXli2不受營(yíng)養(yǎng)狀態(tài)的影響。以上實(shí)驗(yàn)結(jié)果表明ACOXl基因兩種亞型在脊椎動(dòng)物中可能行使不同的功能。
[Abstract]:Oreochromis niloticus (Nile Tilapia) has good growth performance and strong resistance, and has become a world breed. As an important model species, its genome sequencing has been completed, and it is beneficial to carry out the study of fish nutrition physiology with its model. Peroxisome proliferator activated recept (Peroxisome proliferator activated recept) Or gamma, PPARy, a key regulator of animal lipid metabolism, is widely found in vertebrates. However, there is still a lack of research on the physiological function, molecular structure and transcriptional activity of fish PPARy. This study uses Nile tilapia as a model, using molecular cloning, cell culture, fluorescence quantitative PCR, histological analysis, and Western blot. Transcriptional studies, double fluorescein reporting system, promoter capture and metabolite content determination are used to study the following four aspects of PPARy: 1) the role of PPARy in regulating homeostasis of lipid; 2) PPARy structure analysis and transcriptional activity study of tilapia; 3) mammalian activator Rosiglitazone and inhibitor GW9662 The effect of lipid metabolism of tilapia, and 4) the cloning and nutrition regulation of the PPARy target gene ACOX1. The main results and conclusions of this paper are as follows: 1. the natural selection of PPARy in the homeostasis of lipid in Nile tilapia not only endows the energy of the animals when they are rich in lipid, but also endows them with the intake of lipids in the outside world. The ability to synthesize lipids in the body is carried out in the body. However, fish is not well elucidated on how to use its own lipid metabolism system to cope with the different nutritional conditions of low fat and high fat foods. In this study, the two cases of Nile tilapia were investigated by high fat feed and low fat feed. How to maintain the homeostasis in the lipid and what role PPARy plays in this process. The results showed that the growth rate (Growth rate) between the three groups (Hepatic somatic index), the liver body ratio (Hepatic somatic index), the serum, the liver, the muscle and the fat tissues were all content after 10 weeks of feeding of fat and three kinds of feed, respectively. There was no significant difference, but the body fat content and fat body ratio increased with the increase of fat level in the feed. The results of fluorescence quantitative PCR, transcriptional omics, and protein immunoblotting showed that the liver was the main organ to respond to low fat feeding, mainly the increase of glycolysis rate and the enhancement of adipose acid ab initio synthesis ability; in high fat condition. At the same time, the adipose tissue activates PPAR gamma by absorbing more free fatty acids and increasing the hydrolysis of fat, and then activates the activated PPAR gamma to further promote the proliferation of adipocytes, so that the fat cells can store more triglycerides in the same condition. The results show that the PPARy has a similar function to the mammal. The physiological regulation mechanism of fish in response to insufficient and excessive fat intake was systematically clarified, which enriched our understanding of fish physiology,.2. Nile tilapia PPARy structure analysis and transcriptional activity study, the peroxisome proliferator activation receptor y (Peroxisome proliferator activated receptor gamma, PPARy) is animal fat The key regulator of class metabolism, the previous section shows that Nile tilapia PPARy is involved in regulating the proliferation of tilapia adipocyte, and the molecular structure determines its physiological function. On this basis, we further analyzed the molecular structure and transcriptional activity of PPARy. First, the total length of PPARy (NtPPARy) of Nile tilapia was obtained and passed. The transcriptional activity of the NtPPARy and NtRXRa expression vectors and the FABP4 promoter driven report carrier in the HEK-293 cell line was studied. The results showed that there were two transcripts in NtPPARy, the two in the 5 '- non coding region, and the NtPPARy LBD (Ligand binding domain) more than 39 amino acid residues after comparison with the mammalian. The tissue expression pattern showed that the two transcripts showed different distribution patterns in the 11 tissues, but both were highly expressed in the liver, the intestines and the kidneys. The transcriptional activity study showed that NtPPARy and NtRXRa were involved in the transcription of the FABP4 gene of Nile tilapia (Nile Tilapia). The results showed that NtPPARy and NtRXRa were involved in the transcription of the FABP4 gene. In conclusion, the DBD (DNA binding domain) of PPARy is highly conservative, but the LBD conservatism is relatively weak, and the tilapia PPARy and RXRa participate in the transcriptional regulation of the target genes such as FABP4. On the basis of obtaining the Nile tilapia cell line, the plasmid system of this study will be the future. An effective tool for studying fish PPARy function.3. mammalian PPARy activator Rosiglitazone and inhibitor GW9662 on lipid metabolism of tilapia (Gain of function) and functional deletion (Loss of function) are effective strategies for the study of gene function. Functional acquisition and functional deficiency can be quickly realized by using activators and inhibitors. Therefore, we tried to activate and inhibit the PPARy of tilapia by mammalian PPARy activator Rosiglitazone (Rosi) and inhibitor GW9662 (GW9). On this basis, we explored the physiological functions of the tilapia PPARy. This study first designed two kinds of fat level feedstuff, namely, 4% (Standard diet, SD) and 15% (High fat), at each level. Including the control group, the Rosi (15mg/kg) group, and the GW9 (10mg/kg) group, a total of 6 groups of feed were collected and analyzed after 6 weeks of culture. The results showed that the lipid body ratio of the HFD group was significantly higher than that in the SD group. The liver body ratio and the TG content in the liver were also higher than those in the SD group, indicating that the high fat led to the excessive fat deposition in the liver, and the serum TG content of the HFD group was significantly higher than that in the SD group, indicating the homeostasis imbalance in the lipid. We successfully obtained the fatty liver model we needed. However, the addition of Rosi and GW9 had no significant effect on the growth and metabolism of tilapia in low fat or high lipid background. Therefore, two experiments were carried out by intraperitoneal injection of this more direct method. The amount of Rosiglitazone injection was 0 (DMSO), 0.6mg/kg, 18mg/kg and 5. 4 mg/kg; the amount of GW9662 injection was 0 (DMSO), 0.4mg/kg, 12mg/kg, and 36 mg/kg, and the injection of GW9662 on serum triglyceride (Triglyceride, TG), free fatty acids (Free fatty acid), and glucose had no significant effect on blood glucose. TG content increased the content of FFA. Fluorescence quantitative PCR analysis found that fatty acid synthesis in the liver, TG synthesis and VLDL key protein gene expression decreased, indicating that the decrease of TG content was mainly attributed to the decrease of liver lipid synthesis and secretion, and the increase of FFA was attributed to the enhancement of lipid hydrolysis in the muscles. But Rosi did not affect the PPAR gamma in adipose tissue. The expression in the liver and muscle, which regulates the post transcriptional modification of PPAR gamma, affects its protein activity and causes the corresponding physiological effects. The results suggest that short term Rosi injection can promote the cloning of the lipid hydrolysis of the.4. PPAR gamma target gene ACOX1 of the tilapia muscle and its nutritional regulation of peroxisomes. The metabolic activity in peroxisomes is regulated by PPAR gamma, and the ester acyl coenzyme A oxidase 1 (Acayl-CoA Oxidase 1, ACOX1) is the first rate limiting enzyme of peroxisome beta oxidation, and it participates in the catalytic process of ester acyl coenzyme A to 2- CIS enyl coenzyme A, and peroxisome beta oxidation process as a letter. The synthesis of molecule.ACOX1 is one of the target genes of PPAR gamma. Therefore, it is important to elucidate the structure and function of ACOX1 for the study of how PPAR gamma regulates the synthesis of signal molecules of peroxidation objects. In this study, the ACOX1 gene was cloned in Nile tilapia for the first time. It was found to have two transcripts, named ACOXli1, respectively. And ACOXli2, their encoded proteins are composed of 661 amino acid residues. The two subtype of the encoding region is encoded by the exon E3b, which consists of 14 exons, and the ACOXli2, the ACOXli2, is encoded by E3a. The homology analysis shows that the conservatism of ACOXli1 is significantly higher than that of ACOXli2, suggesting that ACOXli1's function may be more than ACOXli2. The mRNA distribution pattern of the two groups showed that the expression of ACOXi1 in the liver was the highest, followed by the kidney and the brain, and the expression of ACOXli2 in the kidney was the highest, followed by the liver. The expression of the two subtypes in the white muscles, gills, brain and kidneys was significantly different, and the ACOXli2 was significantly higher in the white muscles and kidneys than in the ACOXlil, while in the brain, the brain was significantly higher than that in the brain. In contrast to the gills, in the 36 hours of hunger, the nutritional status of 1,3,8,24 hours after full food was found: the two subtypes made the same response pattern in the liver, the kidney ACOXlil and the liver response patterns converged, and the ACOXli2 was not affected by the nutritional status. The experimental results showed that the two subtypes of the ACOXl gene were in the ridge. Different functions may be exercised in vertebroid animals.
【學(xué)位授予單位】：華東師范大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q953
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本文編號(hào)：2174136
[Abstract]:Oreochromis niloticus (Nile Tilapia) has good growth performance and strong resistance, and has become a world breed. As an important model species, its genome sequencing has been completed, and it is beneficial to carry out the study of fish nutrition physiology with its model. Peroxisome proliferator activated recept (Peroxisome proliferator activated recept) Or gamma, PPARy, a key regulator of animal lipid metabolism, is widely found in vertebrates. However, there is still a lack of research on the physiological function, molecular structure and transcriptional activity of fish PPARy. This study uses Nile tilapia as a model, using molecular cloning, cell culture, fluorescence quantitative PCR, histological analysis, and Western blot. Transcriptional studies, double fluorescein reporting system, promoter capture and metabolite content determination are used to study the following four aspects of PPARy: 1) the role of PPARy in regulating homeostasis of lipid; 2) PPARy structure analysis and transcriptional activity study of tilapia; 3) mammalian activator Rosiglitazone and inhibitor GW9662 The effect of lipid metabolism of tilapia, and 4) the cloning and nutrition regulation of the PPARy target gene ACOX1. The main results and conclusions of this paper are as follows: 1. the natural selection of PPARy in the homeostasis of lipid in Nile tilapia not only endows the energy of the animals when they are rich in lipid, but also endows them with the intake of lipids in the outside world. The ability to synthesize lipids in the body is carried out in the body. However, fish is not well elucidated on how to use its own lipid metabolism system to cope with the different nutritional conditions of low fat and high fat foods. In this study, the two cases of Nile tilapia were investigated by high fat feed and low fat feed. How to maintain the homeostasis in the lipid and what role PPARy plays in this process. The results showed that the growth rate (Growth rate) between the three groups (Hepatic somatic index), the liver body ratio (Hepatic somatic index), the serum, the liver, the muscle and the fat tissues were all content after 10 weeks of feeding of fat and three kinds of feed, respectively. There was no significant difference, but the body fat content and fat body ratio increased with the increase of fat level in the feed. The results of fluorescence quantitative PCR, transcriptional omics, and protein immunoblotting showed that the liver was the main organ to respond to low fat feeding, mainly the increase of glycolysis rate and the enhancement of adipose acid ab initio synthesis ability; in high fat condition. At the same time, the adipose tissue activates PPAR gamma by absorbing more free fatty acids and increasing the hydrolysis of fat, and then activates the activated PPAR gamma to further promote the proliferation of adipocytes, so that the fat cells can store more triglycerides in the same condition. The results show that the PPARy has a similar function to the mammal. The physiological regulation mechanism of fish in response to insufficient and excessive fat intake was systematically clarified, which enriched our understanding of fish physiology,.2. Nile tilapia PPARy structure analysis and transcriptional activity study, the peroxisome proliferator activation receptor y (Peroxisome proliferator activated receptor gamma, PPARy) is animal fat The key regulator of class metabolism, the previous section shows that Nile tilapia PPARy is involved in regulating the proliferation of tilapia adipocyte, and the molecular structure determines its physiological function. On this basis, we further analyzed the molecular structure and transcriptional activity of PPARy. First, the total length of PPARy (NtPPARy) of Nile tilapia was obtained and passed. The transcriptional activity of the NtPPARy and NtRXRa expression vectors and the FABP4 promoter driven report carrier in the HEK-293 cell line was studied. The results showed that there were two transcripts in NtPPARy, the two in the 5 '- non coding region, and the NtPPARy LBD (Ligand binding domain) more than 39 amino acid residues after comparison with the mammalian. The tissue expression pattern showed that the two transcripts showed different distribution patterns in the 11 tissues, but both were highly expressed in the liver, the intestines and the kidneys. The transcriptional activity study showed that NtPPARy and NtRXRa were involved in the transcription of the FABP4 gene of Nile tilapia (Nile Tilapia). The results showed that NtPPARy and NtRXRa were involved in the transcription of the FABP4 gene. In conclusion, the DBD (DNA binding domain) of PPARy is highly conservative, but the LBD conservatism is relatively weak, and the tilapia PPARy and RXRa participate in the transcriptional regulation of the target genes such as FABP4. On the basis of obtaining the Nile tilapia cell line, the plasmid system of this study will be the future. An effective tool for studying fish PPARy function.3. mammalian PPARy activator Rosiglitazone and inhibitor GW9662 on lipid metabolism of tilapia (Gain of function) and functional deletion (Loss of function) are effective strategies for the study of gene function. Functional acquisition and functional deficiency can be quickly realized by using activators and inhibitors. Therefore, we tried to activate and inhibit the PPARy of tilapia by mammalian PPARy activator Rosiglitazone (Rosi) and inhibitor GW9662 (GW9). On this basis, we explored the physiological functions of the tilapia PPARy. This study first designed two kinds of fat level feedstuff, namely, 4% (Standard diet, SD) and 15% (High fat), at each level. Including the control group, the Rosi (15mg/kg) group, and the GW9 (10mg/kg) group, a total of 6 groups of feed were collected and analyzed after 6 weeks of culture. The results showed that the lipid body ratio of the HFD group was significantly higher than that in the SD group. The liver body ratio and the TG content in the liver were also higher than those in the SD group, indicating that the high fat led to the excessive fat deposition in the liver, and the serum TG content of the HFD group was significantly higher than that in the SD group, indicating the homeostasis imbalance in the lipid. We successfully obtained the fatty liver model we needed. However, the addition of Rosi and GW9 had no significant effect on the growth and metabolism of tilapia in low fat or high lipid background. Therefore, two experiments were carried out by intraperitoneal injection of this more direct method. The amount of Rosiglitazone injection was 0 (DMSO), 0.6mg/kg, 18mg/kg and 5. 4 mg/kg; the amount of GW9662 injection was 0 (DMSO), 0.4mg/kg, 12mg/kg, and 36 mg/kg, and the injection of GW9662 on serum triglyceride (Triglyceride, TG), free fatty acids (Free fatty acid), and glucose had no significant effect on blood glucose. TG content increased the content of FFA. Fluorescence quantitative PCR analysis found that fatty acid synthesis in the liver, TG synthesis and VLDL key protein gene expression decreased, indicating that the decrease of TG content was mainly attributed to the decrease of liver lipid synthesis and secretion, and the increase of FFA was attributed to the enhancement of lipid hydrolysis in the muscles. But Rosi did not affect the PPAR gamma in adipose tissue. The expression in the liver and muscle, which regulates the post transcriptional modification of PPAR gamma, affects its protein activity and causes the corresponding physiological effects. The results suggest that short term Rosi injection can promote the cloning of the lipid hydrolysis of the.4. PPAR gamma target gene ACOX1 of the tilapia muscle and its nutritional regulation of peroxisomes. The metabolic activity in peroxisomes is regulated by PPAR gamma, and the ester acyl coenzyme A oxidase 1 (Acayl-CoA Oxidase 1, ACOX1) is the first rate limiting enzyme of peroxisome beta oxidation, and it participates in the catalytic process of ester acyl coenzyme A to 2- CIS enyl coenzyme A, and peroxisome beta oxidation process as a letter. The synthesis of molecule.ACOX1 is one of the target genes of PPAR gamma. Therefore, it is important to elucidate the structure and function of ACOX1 for the study of how PPAR gamma regulates the synthesis of signal molecules of peroxidation objects. In this study, the ACOX1 gene was cloned in Nile tilapia for the first time. It was found to have two transcripts, named ACOXli1, respectively. And ACOXli2, their encoded proteins are composed of 661 amino acid residues. The two subtype of the encoding region is encoded by the exon E3b, which consists of 14 exons, and the ACOXli2, the ACOXli2, is encoded by E3a. The homology analysis shows that the conservatism of ACOXli1 is significantly higher than that of ACOXli2, suggesting that ACOXli1's function may be more than ACOXli2. The mRNA distribution pattern of the two groups showed that the expression of ACOXi1 in the liver was the highest, followed by the kidney and the brain, and the expression of ACOXli2 in the kidney was the highest, followed by the liver. The expression of the two subtypes in the white muscles, gills, brain and kidneys was significantly different, and the ACOXli2 was significantly higher in the white muscles and kidneys than in the ACOXlil, while in the brain, the brain was significantly higher than that in the brain. In contrast to the gills, in the 36 hours of hunger, the nutritional status of 1,3,8,24 hours after full food was found: the two subtypes made the same response pattern in the liver, the kidney ACOXlil and the liver response patterns converged, and the ACOXli2 was not affected by the nutritional status. The experimental results showed that the two subtypes of the ACOXl gene were in the ridge. Different functions may be exercised in vertebroid animals.
【學(xué)位授予單位】：華東師范大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q953
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本文編號(hào)：2174136
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