本文選題：共軛亞油酸　切入點(diǎn)：脂肪沉積　出處：《西南大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：共軛亞油酸(Conjugated linoleic acid, CLA)具有抑制動(dòng)物脂肪沉積的作用,但其作用機(jī)制并不完全清楚。本論文結(jié)合動(dòng)物營(yíng)養(yǎng)學(xué)研究手段與分子生物學(xué)技術(shù),從個(gè)體和分子水平系統(tǒng)探討了CLA通過誘導(dǎo)脂肪細(xì)胞凋亡的方式來抑制豬的脂肪沉積,揭示其可能存在的作用機(jī)制。試驗(yàn)選用36頭長(zhǎng)白×榮昌仔豬,體重13.91+1.53kg,隨機(jī)分為3個(gè)處理組,每個(gè)處理組3個(gè)重復(fù),每個(gè)重復(fù)4頭仔豬,分別飼喂含0%(對(duì)照組)、1%和2%CLA的日糧,試驗(yàn)持續(xù)30天。試驗(yàn)結(jié)束時(shí),仔豬空腹12h后稱重,隨后從每個(gè)重復(fù)中選取2頭體重適中的仔豬進(jìn)行靜脈采血,分離得血清,用以血液生化指標(biāo)的測(cè)定。然后,仔豬放血屠宰,測(cè)量背部皮下脂肪厚度,剝離背部脂肪組織和腹部脂肪組織進(jìn)行稱重。分別取大塊背最長(zhǎng)肌和半腱肌,用以肌內(nèi)脂肪含量測(cè)定。此外,每頭仔豬分別取三份脂肪組織,分別用于形態(tài)學(xué)觀察,Caspase-3、-8、-9等凋亡因子酶活分析,以及凋亡相關(guān)因子的基因和蛋白表達(dá)水平檢測(cè)。主要試驗(yàn)結(jié)果如下：1.CLA顯著降低了仔豬日均采食量(P0.05),而對(duì)料重比影響不顯著(P0.05)。1%和2% CLA組分別使仔豬日均增重下降13.13%(P0.05)和7.95%(P0.05)。2.日糧添加CLA顯著減少了仔豬的背部脂肪沉積量(P0.05)。并且隨著CLA添加水平的提高,背膘厚降低。利用生化分析儀檢測(cè)血清,結(jié)果發(fā)現(xiàn)CLA對(duì)仔豬血清中的葡萄糖、甘油三酯、低密度脂蛋白膽固醇和高密度脂蛋白膽固醇等生化指標(biāo)影響不明顯(P0.05),但顯著降低了總膽固醇含量(P0.05)。3.通過對(duì)脂肪組織切片的免疫熒光染色觀察發(fā)現(xiàn)CLA處理組仔豬的背部脂肪細(xì)胞表現(xiàn)出明顯的凋亡跡象,包括染色質(zhì)聚集、核濃縮、DNA斷裂等凋亡特征,尤其是2% CLA組脂肪細(xì)胞凋亡特征更為明顯。酶活檢測(cè)結(jié)果表明日糧添加CLA使仔豬背部脂肪組織中的凋亡因子Caspase-3、-8和-9的活力均顯著升高(P0.05)。熒光定量PCR和western blotting檢測(cè)結(jié)果表明CLA處理下調(diào)了抑凋亡因子Bcl2的mRNA和蛋白表達(dá),升高了促凋亡因子Bax和P53的mRNA及蛋白表達(dá),同時(shí)增加Cyt-C的mRNA表達(dá)并且促進(jìn)Cyt-C蛋白從線粒體釋放到細(xì)胞質(zhì)中。Caspase-3的mRNA表達(dá)上調(diào)和剪切型的Caspase-3蛋白水平的增加說明凋亡執(zhí)行因子Caspase-3被激活并介導(dǎo)了脂肪細(xì)胞凋亡的發(fā)生。另一方面,CLA處理后還提高了脂肪細(xì)胞中TNF-α及其受體TNFR1的mRNA表達(dá)以及蛋白水平,而它們是死亡受體凋亡通路的標(biāo)志因子。以上這些結(jié)果提示線粒體通路和死亡受體通路都參與了CLA誘導(dǎo)的豬背部脂肪細(xì)胞凋亡。以上試驗(yàn)結(jié)果表明,日糧補(bǔ)充CLA能夠通過誘導(dǎo)脂肪細(xì)胞凋亡的方式來減少仔豬的背部脂肪沉沉,且線粒體通路和死亡受體通路都參與了凋亡信號(hào)的傳遞。
[Abstract]:Conjugated linoleic acid (CLA) can inhibit fat deposition in animals, but its mechanism is not completely clear. The mechanism of CLA inhibiting fat deposition in pigs by inducing adipocyte apoptosis was studied systematically at individual and molecular level. 36 Landrace 脳 Rongchang piglets were used in the experiment. The body weight was 13.91 1.53kg, and was randomly divided into three treatment groups, three replicates in each treatment group and four piglets per replica. The piglets were fed diets containing 0% and 2 CLA respectively. The experiment lasted 30 days. At the end of the experiment, the piglets were weighed after 12 hours fasting. Then two moderately weight piglets were selected from each repeat for venous blood collection, and the serum was isolated for the determination of blood biochemical indexes. Then, the piglets were bloodletting and slaughtered, and the thickness of subcutaneous fat on the back was measured. In addition, three portions of fat tissue were taken from each piglet to measure the fat content in the muscle, and the longissimus dorsi muscle and the semitendinosus muscle were taken to measure the intramuscular fat content. It was used to observe the activity of Caspase-3, caspase-3, caspase-8, and other apoptotic factors, respectively. The main results were as follows: 1. CLA significantly reduced the daily feed intake of piglets (P0.05), but had no significant effect on the feed / weight ratio (P0.05). 1% and 2% CLA groups decreased the average daily weight gain of piglets by 13.13 and P0.05, respectively. Dietary supplementation of CLA significantly reduced back fat deposition in piglets and increased the level of CLA supplementation. The serum was detected by biochemical analyzer. The results showed that CLA could treat glucose, triglyceride and triglyceride in serum of piglets. Low density lipoprotein cholesterol (LDL-C) and high density lipoprotein cholesterol (HDL-C) had no obvious effect on the biochemical indexes such as P0.05, but the total cholesterol content was significantly decreased. The immunofluorescence staining of adipose tissue sections showed that the piglets treated with CLA were in the piglets. Fat cells in the back showed obvious signs of apoptosis. Including chromatin aggregation, nuclear condensation DNA fragmentation and other apoptotic characteristics, Especially in 2% CLA group, the apoptotic characteristics of adipocytes were more obvious. The results of enzyme activity test showed that the activities of Caspase-3, Caspase-3 and -9 in dorsal adipose tissue of piglets were significantly increased by adding CLA to diet. Fluorescence quantitative PCR and western blotting were used to detect the apoptosis of adipocytes in piglets. The results showed that CLA treatment down-regulated the expression of mRNA and protein of Bcl2. Increased the expression of mRNA and protein of apoptosis-promoting factor Bax and p53, At the same time, the increase of mRNA expression of Cyt-C and the up-regulation of mRNA expression of Cyt-C protein from mitochondria to cytoplasm. Caspase-3 and the increase of shearing Caspase-3 protein level indicate that Caspase-3 is activated and mediates the apoptosis of adipocytes. On the other hand, the mRNA expression and protein level of TNF- 偽 and its receptor TNFR1 in adipocytes were increased after treatment. These results suggest that both mitochondrial pathway and death receptor pathway are involved in the apoptosis of pig dorsal adipocytes induced by CLA. Dietary supplementation of CLA could reduce back fat accumulation in piglets by inducing adipocyte apoptosis and mitochondrial pathway and death receptor pathway were involved in apoptosis signal transduction.
【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S828.5

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