本文關鍵詞： 肉雞 腹水綜合征 膽固醇代謝 甘油三酯代謝 肝細胞缺氧模型　出處：《中國農業(yè)科學院》2016年碩士論文　論文類型：學位論文

【摘要】：本試驗旨在研究腹水綜合征(Ascites syndrome,AS)肉雞肝臟脂類代謝變化規(guī)律及肝細胞缺氧模型的構建方法。試驗一:選擇1日齡健康羅斯308肉公雞216只,隨機分成正常對照組和低溫誘導組,每組6個重復,每個重復18只雞,試驗期為35天。于21、35日齡隨機從每個重復選取3只雞,檢測AS相關經典指標紅細胞壓積(HCT)、腹水心臟指數(AHI)和腹腔積液,以判定AS模型的構建;測定以上指標后根據AS判定原則,于21、35日齡從正常對照組選取6只健康肉雞設置為對照組,從低溫誘導組選取6只AS肉雞設置為AS組,檢測肝臟總膽固醇(TC)代謝相關基因、蛋白的表達。結果顯示:(1)21 d和35 d時,低溫誘導組肉雞HCT和AHI均極顯著高于正常對照組(P0.01),且腹腔可觀察到黃色積液。(2)21 d時,AS組肉雞血清TC極顯著高于對照組(P0.01),而肝臟TC及35日齡時血清TC、肝臟TC與對照組差異均不顯著。21 d和35 d時,肝臟TC合成相關基因β-羥-β-甲基戊二酸單酰輔酶A還原酶及肝臟TC吸收、轉運相關基因低密度脂蛋白受體、載脂蛋白A I、三磷酸腺苷結合蛋白G5、三磷酸腺苷結合蛋白G8的mRNA水平均差異不顯著;21 d時,肝臟TC分解相關基因膽固醇7α-羥化酶(CYP7A1)和肝X受體α的mRNA表達及CYP7A1蛋白表達顯著降低(P0.05),而35 d時差異不顯著。試驗二:在AS模型構建成功的基礎上,于21、35日齡從正常對照組選取6只健康肉雞設置為對照組,從低溫誘導組選取6只AS肉雞設置為AS組,檢測甘油三酯(TG)變化及其代謝相關基因、蛋白的表達。結果顯示:21 d時,AS組肉雞肝臟TG顯著低于對照組(P0.05),而血清TG及35日齡時血清TG、肝臟TG與對照組差異均不顯著。21 d時,TG合成相關基因脂肪酸合成酶(FAS)、蘋果酸酶(ME)和乙酰輔酶A羧化酶(ACC)的mRNA表達均顯著降低(P0.05),而其轉運相關基因載脂蛋白B(ApoB)m RNA表達及微粒體甘油三酯轉運蛋白(MTP)蛋白表達均差異不顯著(P0.05);35 d時,FAS、ApoB mRNA表達及MTP蛋白表達也顯著降低(P0.05),而ME、ACC m RNA表達差異不顯著(P0.05)。試驗三:以18胚齡雞原代肝細胞為素材,以不同的缺氧條件(氧氣供應為9%、15%)對雞肝細胞進行24 h缺氧培養(yǎng),不同缺氧組均設常氧組作對照,采用CCK-8法檢測細胞活性。隨后檢測缺氧后培養(yǎng)0、2、6、12 h時細胞活性,以摸索不同缺氧條件下肝細胞的缺氧耐受能力。結果顯示:(1)缺氧培養(yǎng)24h,缺氧組(氧氣供應為9%、15%)肝細胞存活率均顯著下降(P0.05)。(2)氧氣供應為9%時,肝細胞存活率下降的時間點在2-6 h之間;氧氣供應為15%時,肝細胞存活率下降的時間點在6-12 h之間。綜上所述,在AS早期,脂類代謝變化更為敏感;AS肉雞TC增加可能是由于TC分解代謝相關基因CYP7A1、LXRα表達下降;AS肉雞TG減少可能是由于TG合成代謝相關基因FAS、ME、ACC表達下降。缺氧會造成雞肝細胞存活率下降,且氧氣供應為9%時下降的時間點在2-6h之間,氧氣供應為15%時下降的時間點在6-12 h之間。
[Abstract]:The purpose of this study was to study Ascites syndrome in ascitic syndrome. Experiment 1: 216 1-day-old Roth308 broilers were randomly divided into two groups: normal control group and hypothermia induction group. The experiment period was 35 days. 3 chickens were randomly selected from each replica at the age of 21 ~ 35 days, and HCT was detected by hematocrit. Ascitic cardiac index (AHI) and peritoneal effusion were used to determine the construction of as model. According to the principle of as determination, 6 healthy broilers from normal control group were selected as control group and 6 as broilers from hypothermia induction group were selected as as group at 21 ~ 35 days of age. The expression of related genes and proteins in liver total cholesterol (TCC) metabolism was detected. The results showed that there were 21 days and 35 days after treatment. HCT and AHI in hypothermia induction group were significantly higher than those in normal control group (P 0.01), and yellow effusion could be observed in abdominal cavity at 21 days. Serum TC in as group was significantly higher than that in control group (P 0.01), but there was no significant difference between liver TC and control group at 21 d and 35 d. Liver TC synthesis related gene 尾 -hydroxy- 尾 -methylglutaric acid monoacyl coA reductase and liver TC absorption, transport associated gene low-density lipoprotein receptor, apolipoprotein A I. The mRNA levels of adenosine triphosphate binding protein G5 and adenosine triphosphate binding protein G8 were not significantly different. At 21 d, the expression of mRNA and CYP7A1 protein of liver TC catabolization-related gene cholesterol 7 偽 -hydroxylase (CYP7A1) and liver X receptor 偽 decreased significantly (P 0.05). However, there was no significant difference at 35 d. Experiment 2: on the basis of the successful construction of as model, 6 healthy broilers were selected from the normal control group as the control group at 21 ~ 35 days of age. Six as broilers were selected from hypothermia induction group to set up as group. The changes of triglyceride (TG) and the expression of metabolism-related genes and proteins were detected. Liver TG in as group was significantly lower than that in control group (P 0.05), while serum TG and TGat 35 days old were not significantly different from those in control group. The mRNA expression of fatty acid synthase (FASA), malate enzyme (MEM) and acetyl coenzyme A carboxylase (ACCs) were significantly decreased (P 0.05). However, there was no significant difference in the expression of apolipoprotein (ApoBm) RNA and microsomal triglyceride transporter (TTP) protein. At 35 days, the expression of ApoB mRNA and MTP protein in FASA was also significantly decreased, while that in ME was significantly decreased. There was no significant difference in the expression of ACC m RNA. Experiment 3: the primary hepatocytes of 18-embryo chicken were used as raw materials, under different hypoxia conditions (oxygen supply was 9%). Chicken hepatocytes were cultured under hypoxia for 24 hours. The normal oxygen group was used as control group and the cell activity was detected by CCK-8 method. At 12 h, the cell activity was studied to explore the hypoxia tolerance ability of hepatocytes under different hypoxia conditions. The results showed that the hypoxia group was cultured for 24 hours (oxygen supply was 9%). When oxygen supply was 9, the survival rate of hepatocytes decreased from 2 to 6 hours. When oxygen supply was 15, the survival rate of hepatocytes decreased from 6 to 12 hours. In conclusion, the changes of lipid metabolism were more sensitive at the early stage of as. The increase of TC in as broilers may be due to the decrease of LXR 偽 expression of CYP7A1 gene associated with TC catabolism. The decrease of TG in as broilers may be due to the decrease in the expression of FASMEC ACC, which is related to the synthesis of TG, and hypoxia may lead to the decrease of survival rate of chicken hepatocytes. When oxygen supply is 9, the time point is between 2 and 6 hours, and the oxygen supply at 15 is between 6 and 12 hours.
【學位授予單位】：中國農業(yè)科學院
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：S858.31

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