腹水综合征肉鸡肝脏脂类代谢研究与细胞模型构建
本文关键词: 肉鸡 腹水综合征 胆固醇代谢 甘油三酯代谢 肝细胞缺氧模型 出处:《中国农业科学院》2016年硕士论文 论文类型:学位论文
【摘要】:本试验旨在研究腹水综合征(Ascites syndrome,AS)肉鸡肝脏脂类代谢变化规律及肝细胞缺氧模型的构建方法。试验一:选择1日龄健康罗斯308肉公鸡216只,随机分成正常对照组和低温诱导组,每组6个重复,每个重复18只鸡,试验期为35天。于21、35日龄随机从每个重复选取3只鸡,检测AS相关经典指标红细胞压积(HCT)、腹水心脏指数(AHI)和腹腔积液,以判定AS模型的构建;测定以上指标后根据AS判定原则,于21、35日龄从正常对照组选取6只健康肉鸡设置为对照组,从低温诱导组选取6只AS肉鸡设置为AS组,检测肝脏总胆固醇(TC)代谢相关基因、蛋白的表达。结果显示:(1)21 d和35 d时,低温诱导组肉鸡HCT和AHI均极显著高于正常对照组(P0.01),且腹腔可观察到黄色积液。(2)21 d时,AS组肉鸡血清TC极显著高于对照组(P0.01),而肝脏TC及35日龄时血清TC、肝脏TC与对照组差异均不显著。21 d和35 d时,肝脏TC合成相关基因β-羟-β-甲基戊二酸单酰辅酶A还原酶及肝脏TC吸收、转运相关基因低密度脂蛋白受体、载脂蛋白A I、三磷酸腺苷结合蛋白G5、三磷酸腺苷结合蛋白G8的mRNA水平均差异不显著;21 d时,肝脏TC分解相关基因胆固醇7α-羟化酶(CYP7A1)和肝X受体α的mRNA表达及CYP7A1蛋白表达显著降低(P0.05),而35 d时差异不显著。试验二:在AS模型构建成功的基础上,于21、35日龄从正常对照组选取6只健康肉鸡设置为对照组,从低温诱导组选取6只AS肉鸡设置为AS组,检测甘油三酯(TG)变化及其代谢相关基因、蛋白的表达。结果显示:21 d时,AS组肉鸡肝脏TG显著低于对照组(P0.05),而血清TG及35日龄时血清TG、肝脏TG与对照组差异均不显著。21 d时,TG合成相关基因脂肪酸合成酶(FAS)、苹果酸酶(ME)和乙酰辅酶A羧化酶(ACC)的mRNA表达均显著降低(P0.05),而其转运相关基因载脂蛋白B(ApoB)m RNA表达及微粒体甘油三酯转运蛋白(MTP)蛋白表达均差异不显著(P0.05);35 d时,FAS、ApoB mRNA表达及MTP蛋白表达也显著降低(P0.05),而ME、ACC m RNA表达差异不显著(P0.05)。试验三:以18胚龄鸡原代肝细胞为素材,以不同的缺氧条件(氧气供应为9%、15%)对鸡肝细胞进行24 h缺氧培养,不同缺氧组均设常氧组作对照,采用CCK-8法检测细胞活性。随后检测缺氧后培养0、2、6、12 h时细胞活性,以摸索不同缺氧条件下肝细胞的缺氧耐受能力。结果显示:(1)缺氧培养24h,缺氧组(氧气供应为9%、15%)肝细胞存活率均显著下降(P0.05)。(2)氧气供应为9%时,肝细胞存活率下降的时间点在2-6 h之间;氧气供应为15%时,肝细胞存活率下降的时间点在6-12 h之间。综上所述,在AS早期,脂类代谢变化更为敏感;AS肉鸡TC增加可能是由于TC分解代谢相关基因CYP7A1、LXRα表达下降;AS肉鸡TG减少可能是由于TG合成代谢相关基因FAS、ME、ACC表达下降。缺氧会造成鸡肝细胞存活率下降,且氧气供应为9%时下降的时间点在2-6h之间,氧气供应为15%时下降的时间点在6-12 h之间。
[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 伪 -hydroxylase (CYP7A1) and liver X receptor 伪 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 伪 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.
【学位授予单位】:中国农业科学院
【学位级别】:硕士
【学位授予年份】:2016
【分类号】:S858.31
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