摘要
为揭示PPARα基因对骡鸭肝细胞PPAR信号通路中脂质代谢基因的调控作用,试验以16日胚龄的骡鸭为材料,用Ⅳ型胶原酶法分离原代肝细胞,分离的原代肝细胞经形态学观察、糖原染色和白蛋白(ALB)检测鉴定后用于后续试验;构建4对干扰PPARα基因的siRNA,分别转染骡鸭原代肝细胞,转染12 h后用荧光显微镜检测转染效率,转染48 h后用油红O染色检测肝细胞脂质分泌情况,筛选干扰效率最高的siRNA,qRT-PCR检测该siRNA干扰PPARα基因后PPAR信号通路上脂质代谢相关基因的表达情况。结果显示:Ⅳ型胶原酶法分离的骡鸭原代肝细胞活性较好,ALB的分泌量维持在较高水平,糖原染色发现大量细胞存在双核和多核状态,干扰效率最高的PPARα-1344组与对照组相比脂质分泌量减少,原代肝细胞中LPL、FABP、FAS和ACBP基因表达量极显著下调(P<0.01),SCD基因表达量显著下调(P<0.05),APO-A1、CYP7A1和CPT1基因表达量差异不显著(P>0.05)。研究表明:分离的骡鸭原代肝细胞纯度好、活性高,PPARα基因可能促进脂肪在肝脏中沉积,与骡鸭的肥肝形成密切相关。
This experiment aimed to reveal the regulatory effect of PPARα gene on lipid metabolism-related genes in the PPAR signaling pathway of hepatocytes of mule duck. The primary hepatocytes were isolated from 16-day mule duck embryos using type Ⅳ collagenase. The isolated primary hepatocytes were identified by morphological observation,glycogen staining and albumin(ALB) detection, and used for subsequent experiments. Four pairs of siRNA were constructed to interfere with PPARα gene. The primary mule duck hepatocytes were transfected, respectively. After transfection for 12 h, the transfection efficiency was detected by fluorescence microscopy. After transfection for 48 h,the lipid secretion of hepatocytes was detected by oil red O staining, and the group with the highest interference efficiency was selected. The expression of lipid metabolism-related genes in the PPAR signaling pathway was determined using qRT-PCR. The results showed that the primary hepatocytes of mule duck isolated with type Ⅳ collagenase had high activity, ALB secretion was maintained at a high level, and glycogen staining revealed binuclear and polynuclear states in a large number of cells. The PPARα-1344 group with the highest interference efficiency had a lower lipid secretion compared with the control group, and the expression of LPL, FABP, FAS and ACBP genes was very significantly down-regulated(P<0.01), the expression of SCD gene was significantly down-regulated(P<0.05), and the expression of APO-A1, CYP7 A1 and CPT1 genes was not significantly different(P>0.05). The results demonstrated that the primary hepatocytes of mule duck isolated in this experiment had high purity and activity, and PPARα gene might promote the deposition of fat in liver, which was closely related to the formation of fatty liver in mule duck.
作者
蓝立明
潘晓丽
何文峰
白丁平
张福君
郑永干
李昂
LAN Liming;PAN Xiaoli;HE Wenfeng;BAI Dingping;ZHANG Fujun;ZHENG Yonggan;LI Ang(College of Animal Science(College of Bee Science),Fujian Agriculture and Forestry University,Fuzhou,Fujian 350002;Jilin Zhengfang Agriculture and Animal Husbandry Co.,Ltd.,Meihekou,Jilin 135000)
出处
《中国家禽》
北大核心
2020年第7期6-12,共7页
China Poultry
基金
现代农业产业技术体系建设专项资金(CARS-43-5)。
