本文關(guān)鍵詞：EPA及其前體ALA對(duì)草魚脂肪蓄積的調(diào)控作用及機(jī)制分析　出處：《西北農(nóng)林科技大學(xué)》2017年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 草魚 脂肪蓄積 EPA ALA

【摘要】：脂肪組織被認(rèn)為是機(jī)體最大的能量儲(chǔ)存器官,同時(shí)也是機(jī)體一個(gè)重要的內(nèi)分泌器官。其在能量平衡、動(dòng)物繁殖、器官保護(hù)、性別選擇等多個(gè)方面具有重要作用。當(dāng)前,隨著魚粉、魚油資源量的下降和價(jià)格的不斷攀升,大豆油、紅花油、葵花籽油等富含n-6系列脂肪酸而相對(duì)缺乏n-3系列脂肪酸的植物性油脂源被廣泛應(yīng)用于草魚的商業(yè)飼料中。除此之外,為了應(yīng)對(duì)巨大的商業(yè)壓力,高能商業(yè)飼料也廣泛被使用以促進(jìn)養(yǎng)殖魚類的生長速度,加速其上市時(shí)間。然而,高能以及n-6/n-3比率失衡的日糧極易導(dǎo)致養(yǎng)殖魚類脂肪的過度蓄積,引發(fā)諸多問題,如死亡率上升、攝食量下降、免疫力下降、品質(zhì)降低等,最終影響經(jīng)濟(jì)效益。因此,對(duì)脂肪過度蓄積的調(diào)控顯得尤為重要。目前,已有學(xué)者通過在體與離體兩種模型,對(duì)脂肪蓄積的調(diào)控進(jìn)行了一定的研究,發(fā)現(xiàn)二十碳五烯酸(Eicosapentaenoic acid,EPA)可以通過促進(jìn)脂肪細(xì)胞凋亡、抑制脂肪合成等途徑降低脂肪蓄積,但其具體機(jī)制尚不明確。Alpha-亞麻酸(Linolenic acid,ALA)是重要的n-3 PUFAs(polyunsaturated fatty acids)之一,也是草魚的必需脂肪酸之一。有研究指出,日糧中添加一定量的ALA可以降低脂肪蓄積。鑒于EPA有限的來源而導(dǎo)致的使用限制,以及草魚具有將C18的ALA轉(zhuǎn)化成EPA的能力,本論文旨在探討EPA及其前體ALA這兩種重要n-3 PUFAs對(duì)草魚脂肪蓄積調(diào)控的作用及機(jī)制,以期為淡水魚類脂質(zhì)代謝提供一定的理論參考。主要研究結(jié)果如下:1.EPA影響草魚腹腔脂肪組織脂質(zhì)蓄積的在體與離體研究。采用EPA的在體飼喂,結(jié)合草魚脂肪細(xì)胞的離體培養(yǎng),對(duì)試驗(yàn)魚生長、生物學(xué)性狀、血清生化、脂肪酸組成等進(jìn)行評(píng)估,并采用HE染色、RT-PCR、流式細(xì)胞術(shù)等檢測方法確定了EPA對(duì)草魚脂肪蓄積的具體作用及機(jī)制。結(jié)果表明:用富含0.52%EPA的日糧飼喂草魚,其生長并未受到影響。經(jīng)過3周的飼養(yǎng),試驗(yàn)魚的腹腔脂肪指數(shù)顯著降低,而當(dāng)飼養(yǎng)時(shí)間延長到6周,腹腔脂肪指數(shù)無顯著差異。同時(shí),在體和離體結(jié)果均顯示EPA顯著促進(jìn)了脂肪組織/細(xì)胞脂肪組織甘油三酯脂酶(ATGL)、激素敏感性脂酶(HSL)-a、過氧化物酶體增殖激活物受體(PPAR)-γ、脂蛋白脂酶(LPL)、caspase 3a、caspase 3b的mRNA水平以及caspase 3的酶活性,下調(diào)了肝X受體(LXR)-α、固醇調(diào)節(jié)原件結(jié)合蛋白(SREBP)-1c、脂肪酸合成酶(FAS)的基因表達(dá)以及Bcl-2/Bax的比率。離體結(jié)果表明,EPA以劑量和時(shí)間依賴型的方式抑制草魚前體脂肪細(xì)胞的活力,且當(dāng)其濃度達(dá)到650μM,作用4天時(shí),處理組細(xì)胞活力只有對(duì)照組的約50%。此時(shí),流式細(xì)胞術(shù)檢測凋亡的結(jié)果顯示,處理組凋亡率高達(dá)37%,顯著高于對(duì)照組。且ROS含量也顯著上升。而抗氧化劑α-生育酚的使用可顯著緩解EPA誘導(dǎo)的凋亡。另一方面,EPA的使用可顯著促進(jìn)脂肪組織/細(xì)胞的EPA含量。2.epa促進(jìn)草魚脂肪細(xì)胞表達(dá)甘油激酶基因。根據(jù)試驗(yàn)一的研究結(jié)果,為深入研究epa短期飼喂能降低脂肪含量,而長期飼喂則無此作用的內(nèi)在機(jī)制。首先,克隆了草魚甘油激酶(gyk)基因部分序列,發(fā)現(xiàn)其在肝胰臟、脂肪組織和腎臟中表達(dá)最高。隨著脂肪細(xì)胞的發(fā)現(xiàn),其表達(dá)呈現(xiàn)上升的趨勢。用100μm的epa處理細(xì)胞6、12、24h,發(fā)現(xiàn)其能顯著促進(jìn)gyk的基因表達(dá)和酶活性,同時(shí)atgl和pparγ的基因表達(dá)也顯著上調(diào)。為進(jìn)一步探究epa誘導(dǎo)草魚脂肪細(xì)胞表達(dá)gyk基因的潛在機(jī)制,分別以atgl和pparγ的抑制劑hy-15859、gw9662孵育細(xì)胞,結(jié)果顯示此兩種抑制劑均能減弱epa對(duì)gyk的誘導(dǎo)作用。此外,對(duì)細(xì)胞進(jìn)行饑餓24h處理,雖然atgl顯著上調(diào),但gyk的酶活性并未發(fā)生變化。而包括α-亞麻酸(18:3n-3,ala)、二十二碳六烯酸(22:6n-3,dha)、亞油酸(18:2n-6,lna)在內(nèi)的pparγ的其余配體脂肪酸,均能促進(jìn)gyk的基因表達(dá)和酶活性。3.日糧添加α-亞麻酸對(duì)草魚稚魚脂質(zhì)代謝,脂肪酸組成及腹腔脂肪細(xì)胞凋亡的影響。分別用富含0.0%、1.0%、2.0%ala的日糧飼喂草魚稚魚8周。結(jié)果發(fā)現(xiàn),日糧中添加ala對(duì)試驗(yàn)草魚的生長無負(fù)面影響。同時(shí),腹腔脂肪指數(shù)在2.0%組最低,而caspase3、caspase8、caspase9的酶活性此組最高。與其他兩組相比,2.0%的ala顯著下調(diào)了fas、srebp-1c的基因表達(dá)以及bcl-2/bax的比率。同時(shí),2.0%組試驗(yàn)魚腹腔脂肪組織出現(xiàn)更多的凋亡樣線粒體,主要表現(xiàn)為線粒體腫脹、嵴消失等。另一方面,對(duì)脂肪酸生物合成途徑的基因表達(dá)檢測發(fā)現(xiàn),肌肉和肝胰臟中,ala組脂肪酸延長酶(elo)和脂肪酸去飽和酶(fad)的表達(dá)顯著高于對(duì)照組,而在腹腔脂肪組織中也呈現(xiàn)出相反的趨勢。然而,日糧中添加ala均能顯著促進(jìn)肌肉、肝胰臟和腹腔脂肪組織的epa和dha的含量。4.α-亞麻酸對(duì)草魚脂肪細(xì)胞凋亡的影響及其機(jī)制分析。利用草魚離體脂肪細(xì)胞培養(yǎng)平臺(tái),用ala分別對(duì)草魚前體以及成熟脂肪細(xì)胞進(jìn)行處理。結(jié)果表明,ala(0-800μm)并未對(duì)細(xì)胞活力產(chǎn)生抑制作用,且當(dāng)處理時(shí)間為2和4天時(shí),ala促進(jìn)細(xì)胞活力。此外,不同分化階段的前體細(xì)胞具有相同的應(yīng)答模式。分別用100、650、1300μm的ala處理前體細(xì)胞2和4天,隨著ala濃度的升高,caspase3a、caspase3b和caspase8的mrna水平顯著下降,而bcl-2/bax則呈現(xiàn)出相反的變化趨勢。而當(dāng)處理時(shí)間延長到8天,caspase9、caspase3b和caspase8的mrna水平在1300μm組最高,而bcl-2/bax在此組最低。對(duì)于成熟脂肪細(xì)胞,經(jīng)過2天的處理,隨著ala濃度的升高,caspase3a、caspase3b、caspase8和caspase9的基因表達(dá)顯著下調(diào),而bcl-2/bax則呈現(xiàn)出相反的變化趨勢。然而,經(jīng)過8天的處理,caspase3a、caspase3b、caspase8和caspase9的基因表達(dá)顯著上調(diào),而bcl-2/bax則呈現(xiàn)出先上升后下降的趨勢,且最低值出現(xiàn)在1300μm組。av/pi凋亡染色結(jié)果也表明,當(dāng)處理時(shí)間為2和4天時(shí),ala主要表現(xiàn)為抗凋亡,而當(dāng)處理時(shí)間延長到8天,650和1300μm組的凋亡率顯著高于其他兩組。對(duì)于脂肪酸組成的檢測發(fā)現(xiàn),用1300μm的ala處理8天,前體脂肪細(xì)胞處理組的ala和dha含量顯著高于對(duì)照組,而成熟脂肪細(xì)胞處理組只有ALA的含量顯著高于對(duì)照組。綜上所述,1)日糧中添加適量的EPA在一定條件下可以通過促進(jìn)甘油三酯水解、促進(jìn)脂肪細(xì)胞凋亡、抑制脂肪酸從頭合成等途徑降低草魚腹腔脂肪蓄積;2)不同于哺乳動(dòng)物,草魚脂肪組織可以表達(dá)GyK基因。EPA通過誘導(dǎo)GyK的表達(dá),促進(jìn)甘油三酯的重新酯化,從而出現(xiàn)補(bǔ)償性合成。且EPA對(duì)GyK的誘導(dǎo)受到PPARγ的調(diào)控,ATGL也參與調(diào)控這一過程;3)日糧中2.0%的ALA可以過促進(jìn)脂肪細(xì)胞凋亡、抑制脂肪酸的從頭合成等途徑降低草魚腹腔脂肪蓄積。且ALA的添加可以顯著促進(jìn)魚體肌肉n-3長鏈多不飽和脂肪酸的沉積,從而提高魚體的品質(zhì);4)ALA本身可以促進(jìn)成熟脂肪細(xì)胞凋亡,而對(duì)于前體脂肪細(xì)胞,ALA可能通過其生物合成產(chǎn)物發(fā)揮作用。草魚前體和成熟脂肪細(xì)胞在利用ALA合成DHA的能力上可能具有差異。
[Abstract]:Adipose tissue is considered to be the biggest energy storage organ, the body is also an important endocrine organ. The balance of energy, animal breeding, protection of organs, plays an important role in the aspects of gender selection. At present, with fish meal, fish oil resources volume decline and prices rising, soybean oil, safflower oil. Sunflower seed oil rich in n-6 fatty acids and relative lack of commercial feed n-3 series of fatty acids in vegetable oil source is widely used in grass carp. In addition, in order to cope with the huge commercial pressure, high commercial feed is also widely used to promote the growth of farmed fish, accelerate their time to market. However, dietary energy and n-6/n-3 ratio imbalance can easily lead to excessive accumulation of fat fish, causing many problems, such as increased mortality, decreased food intake, decreased immunity, reducing the quality of the most. It affects economic benefits. Therefore, to control excessive accumulation of fat is particularly important. At present, some scholars through in vivo and in vitro two models, regulation of fat accumulation was studied, found that twenty of five carbon acid (Eicosapentaenoic acid, EPA) through promoting fat cell apoptosis, inhibit fat synthesis other ways to reduce fat accumulation, but the specific mechanism is not clear.Alpha- linolenic acid (Linolenic acid ALA) is an important n-3 PUFAs (polyunsaturated fatty acids) is one of the essential fatty acids is one of grass carp. Studies have shown that ALA was added in the diet can reduce fat accumulation. In view of the restriction of the use of source EPA due to the limited, and grass carp has the C18 ALA into EPA, this thesis aims to discuss the EPA and its precursor ALA these two important n-3 PUFAs regulation of fat accumulation and the role of grass carp The mechanism, in order to provide a theoretical reference for freshwater fish lipid metabolism. The main results are as follows: the accumulation effect of 1.EPA grass carp abdominal adipose tissue lipid in vivo and in vitro studies. Using EPA in vitro were fed with grass carp fat cells, the test fish growth, biological traits, serum biochemical. The fatty acid composition and evaluated by HE staining, RT-PCR, flow cytometry detection method to determine the specific role and mechanism of EPA on grass carp lipopexia. The results showed that: with the 0.52%EPA rich diet of grass carp, its growth is not affected. After 3 weeks of feeding, fish abdominal fat index test significantly, when the feeding time is prolonged to 6 weeks, there was no significant difference between abdominal fat index. At the same time, in vivo and in vitro results showed that EPA significantly promoted the adipose tissue / cell adipose triglyceride lipase (ATGL), hormone Sensitive lipase (HSL) -a, peroxisome proliferator activated receptor gamma (PPAR), lipoprotein lipase (LPL), caspase 3a, caspase 3B enzyme activity level of mRNA and caspase 3, reduced liver X receptor (LXR) alpha, sterol regulatory element binding protein (SREBP) -1c, fatty acid synthase (FAS) gene expression and Bcl-2/Bax ratio. In vitro results show that grass carp preadipocyte cell viability inhibition of EPA in a dose and time dependent manner, and when the concentration reached 650 M, 4 days, about 50%.. The cell viability in the control group only at this time, flow type detection of apoptosis FCM results showed that the apoptosis rate of treatment group reached 37%, significantly higher than the control group. And the content of ROS also increased significantly. The use of antioxidants, tocopherol can significantly relieve the apoptosis induced by EPA. On the other hand, the use of EPA can significantly promote the.2.epa EPA content of adipose tissue / cells Grass carp to promote fat cell expression of glycerol kinase gene. According to the results of a test, for further research on EPA short-term feeding can reduce the fat content, the intrinsic mechanism and long-term feeding is no effect. First, cloning grass carp glycerol kinase (gyk) gene, found in hepatopancreas, the highest expression of adipose tissue and kidney. As the fat cells found that its expression showed a rising trend. EPA cells treated with 6,12,24h for 100 m, it can significantly promote gene expression and enzyme activity of gyk, ATGL and PPAR gamma gene expression was also up-regulated. To further explore the potential mechanisms for EPA induced grass carp fat cells gyk gene expression, respectively by ATGL and PPAR gamma inhibitor hy-15859, GW9662 cells were incubated. The results show that the two inhibitors can reduce the induction effect of EPA on gyk. In addition, the cells were hungry at 24h, while ATGL A significant increase of the gyk activity did not change. Including alpha linolenic acid (18:3n-3, ALA), twenty-two carbon (22:6n-3, DHA) six acid, linoleic acid (18:2n-6, LNA), PPAR gamma ligand remaining fatty acids, can promote the gyk gene expression and enzyme activity of.3. diet add alpha linolenic acid on the metabolism of juvenile grass carp lipid, fatty acid composition and apoptosis of peritoneal fat cells were used with 0%, 1%, 2.0%ala diet for 8 weeks. The results showed that the larvae of grass carp, no negative effects of ALA supplementation on the growth of grass carp test. At the same time, abdominal fat index in 2% groups the lowest, while Caspase3, caspase8, caspase9 activity of this group was the highest. Compared with the other two groups, 2% ala decreased FAS, SREBP-1c gene expression and bcl-2/bax ratio. At the same time, 2% groups of experimental fish abdominal adipose tissue mitochondria appear more apoptosis, mainly Mitochondrial swelling, cristae disappeared. On the other hand, detected expression of fatty acid biosynthesis pathway genes, muscle and hepatopancreas of group ala, fatty acid elongase (ELO) and fatty acid desaturase (FAD) expression was significantly higher than the control group, while in the abdominal adipose tissue is also showing on the contrary trend. However, dietary supplementation of ALA could significantly promote the muscle, liver and pancreas and abdominal adipose tissue of the EPA and DHA.4. content of alpha linolenic acid on apoptosis of adipocytes and its mechanism. The grass carp platform in vitro fat cells by grass carp, grass carp respectively with ALA precursor and mature fat cells were treated. The results showed that ALA (0-800 m) did not have inhibitory effect on cell viability, and when the treatment time was 2 and 4 days, ALA promotes cell viability. In addition, the same should be a model of progenitor cells with different differentiation stages. By 100, Ala 6501300 m precursor cells 2 and 4 days, with the increase of ALA concentration, caspase3a, caspase3b and caspase8 mRNA were significantly decreased, while bcl-2/bax decreased. When the treatment time was prolonged to 8 days, caspase9, mRNA caspase3b and caspase8 of the water level was highest in 1300 m in this group, and bcl-2/bax group was the lowest. The mature fat cells, after 2 days treatment, with the increase of the concentration of ala, caspase3a, caspase3b, caspase8 and caspase9 gene expression were significantly reduced, while bcl-2/bax decreased. However, after 8 days of treatment, caspase3a, caspase3b, caspase8 and caspase9 gene expression was significantly up-regulated, while bcl-2/bax showed a downward trend after the first rise, and the lowest value appeared in 1300 m group,.Av/pi staining results also show that when the treatment time was 2 and 4 days, ALA mainly for anti apoptosis, and When the treatment time was extended to 8 days, 650 and 1300 m group, the apoptosis rate was significantly higher than that in other two groups. For the detection of fatty acid composition, with 1300 m ala for 8 days, ALA and DHA in preadipocytes treated group was significantly higher than the control group, the content of mature fat cells in treatment group only ALA was significantly higher than the control group. In summary, 1) EPA addition in diets can promote triglyceride hydrolysis under certain conditions, promote fat cell apoptosis, inhibition of fatty acid biosynthesis and other ways to reduce abdominal fat accumulation grass carp; 2) different from the mammalian animal, grass carp can adipose tissue expression of GyK gene by.EPA the expression of GyK, promote the re esterification of triglycerides, and compensatory synthesis. The induction of GyK and EPA is regulated by PPAR gamma, ATGL is also involved in the regulation of this process; 3) the 2% diets can promote fat cells ALA Apoptosis, inhibition of de novo synthesis of fatty acids and other ways to reduce abdominal fat accumulation and grass carp. The addition of ALA can significantly promote the deposition of fish muscle n-3 long chain polyunsaturated fatty acids, thereby improving the quality of fish body; 4) ALA itself can promote the mature adipose cell apoptosis in preadipocytes, ALA may play a role in the biosynthesis of the product. The precursor and mature fat cells may have differences in the ability of using grass carp ALA DHA synthesis.

【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：S917.4

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1 茅達(dá)球;施巧琴;吳松剛;;綠色巴夫藻的EPA發(fā)酵特性研究[A];中國生物化學(xué)與分子生物學(xué)會(huì)第八屆會(huì)員代表大會(huì)暨全國學(xué)術(shù)會(huì)議論文摘要集[C];2001年

2 魏東;張學(xué)成;;富含EPA的海洋微藻眼點(diǎn)擬微球藻的大規(guī)模培養(yǎng)[A];中國藻類學(xué)會(huì)第十一次學(xué)術(shù)討論會(huì)論文摘要集[C];2001年

3 蔡文潔;;EPA全面禁用氫氯氟碳[A];第27屆中國氣象學(xué)會(huì)年會(huì)大氣物理學(xué)與大氣環(huán)境分會(huì)場論文集[C];2010年

4 魏東;張學(xué)成;;抗除草劑海洋微藻的生長和積累EPA的特性[A];中國藻類學(xué)會(huì)第十一次學(xué)術(shù)討論會(huì)論文摘要集[C];2001年

5 劉偉民;馬海樂;李國文;;魚油生理活性物質(zhì)EPA和DHA分離進(jìn)展[A];中國西部農(nóng)產(chǎn)品加工及產(chǎn)業(yè)化發(fā)展戰(zhàn)略研討會(huì)論文集[C];2001年

6 劉偉民;馬海樂;李國文;;超臨界CO_2連續(xù)濃縮魚油EPA和DHA初探[A];中國西部農(nóng)產(chǎn)品加工及產(chǎn)業(yè)化發(fā)展戰(zhàn)略研討會(huì)論文集[C];2001年

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1 梁寶衛(wèi)　編譯;EPA談判,日本有什么牌可打[N];上海證券報(bào);2012年

2 記者 李浩;日本與歐盟將開啟EPA談判[N];金融時(shí)報(bào);2013年

3 北京商報(bào)記者 韓哲 趙毅波;日本發(fā)力EPA戰(zhàn)略的圖謀[N];北京商報(bào);2014年

4 中國現(xiàn)代國際關(guān)系研究院日本所副研究員 劉軍紅;日本EPA框定東盟[N];東方早報(bào);2007年

5 實(shí)習(xí)記者 徐巖;EPA將推出新殺蟲劑標(biāo)簽[N];中國化工報(bào);2009年

6 薛飛;現(xiàn)場總線標(biāo)準(zhǔn)EPA成為國際標(biāo)準(zhǔn)[N];中國知識(shí)產(chǎn)權(quán)報(bào);2007年

7 ;讓你的EPA圖標(biāo)動(dòng)起來[N];中國電腦教育報(bào);2002年

8 龐曉華;EPA出臺(tái)法規(guī)減少柴油污染[N];中國石油報(bào);2004年

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1 雷彩霞;EPA及其前體ALA對(duì)草魚脂肪蓄積的調(diào)控作用及機(jī)制分析[D];西北農(nóng)林科技大學(xué);2017年

2 章涵;EPA實(shí)時(shí)可靠通信協(xié)同調(diào)度與優(yōu)化研究[D];浙江大學(xué);2010年

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1 徐登輝;卵磷脂、膽固醇、EPA和DHA對(duì)團(tuán)頭魴（Megalobrama amblycephala）生長、生理的影響[D];蘇州大學(xué);2015年

2 蔣萬珊;EPA實(shí)時(shí)以太網(wǎng)技術(shù)在智能變電站中的應(yīng)用研究[D];云南大學(xué);2015年

3 朱麗杰;金槍魚油中DHA和EPA富集方法的研究[D];河南工業(yè)大學(xué);2016年

4 劉芳;酶法催化制備富含EPA和DHA甘油三酯研究[D];暨南大學(xué);2016年

5 陳靖;玉米幼嫩葉段再生體系的建立及EPA在玉米中的異源合成[D];山東農(nóng)業(yè)大學(xué);2011年

6 桂麗娟;EPA合成相關(guān)酶基因?qū)γ藁ǖ倪z傳轉(zhuǎn)化[D];山東農(nóng)業(yè)大學(xué);2013年

7 易明華;EPA協(xié)議一致性測試系統(tǒng)的研究與開發(fā)[D];重慶郵電大學(xué);2006年

8 陳鵬;EPA網(wǎng)絡(luò)芯片驗(yàn)證平臺(tái)的設(shè)計(jì)與實(shí)現(xiàn)[D];浙江大學(xué);2011年

9 賈凱麗;EPA工業(yè)以太網(wǎng)監(jiān)控組態(tài)軟件的研究與設(shè)計(jì)[D];浙江大學(xué);2011年

10 班偉;EPA工業(yè)現(xiàn)場設(shè)備管理技術(shù)的研究與實(shí)現(xiàn)[D];大連理工大學(xué);2007年

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