油酸诱导鸡胚原代肝细胞脂沉积模型的构建

doi:10.3864/j.issn.0578-1752.2024.23.017

摘要/Abstract

摘要：

【背景】脂肪肝综合征（fatty liver syndrome，FLS）是蛋鸡产蛋期常见的营养代谢性疾病，包括“简单脂肪变性-脂肪性肝炎-肝纤维化-肝硬化”等多阶段发病过程，给我国家禽产业带来一定经济损失。脂肪肝模型构建是探究FLS发病机理，筛选营养调控措施及后续疾病预防评价的关键。然而，关于家禽肝细胞脂肪变性模型的研究较少。【目的】以鸡胚原代肝细胞（chicken embryo liver，CEL）为研究对象，通过油酸诱导建立原代肝细胞脂沉积模型，并探究FLS不同阶段所需的诱导时间，为家禽FLS的研究提供理论依据。【方法】选用18胚龄的CEL，待细胞生长至铺满皿底80%左右，更换含400 μmol·L^-1油酸的培养基进行处理，培养24、48和72 h后，对细胞进行油红O染色，并测定脂质含量，同时分析与脂代谢、炎症及纤维化相关基因的mRNA和蛋白水平。【结果】与对照组相比，油酸处理24 h后细胞开始出现大量红色脂滴，且随诱导时间的增加红色逐渐加深（P<0.05），表明细胞内脂滴增多，同时TC、TG水平显著升高（P<0.05）。RT-PCR结果显示，油酸诱导24 h后，ACC、FAS、ELOVL6、SREBP等脂合成相关基因表达量显著升高（P<0.05），表明油酸处理24 h后细胞发生脂沉积。ELISA结果显示，IL-6、IL-1β和TNF-α含量在油酸诱导24 h时无显著变化（P>0.05），随诱导时间增加，IL-6水平在48 h时有升高趋势（P=0.052），而在油酸处理72 h后，IL-6和IL-1β水平都显著高于对照组（P<0.05），表明油酸处理48 h后细胞开始发生炎症反应并随诱导时间延长而增加。此外，Western blot结果显示，与对照组相比，48 h油酸处理组IL-6蛋白表达水平显著升高（P<0.05），与ELISA结果一致。同时促纤维化标志物COL1A1和FIBRONECTIN蛋白表达水平升高（P<0.05）；而72 h油酸处理组的IL-6、COL1A1和FIBRONECTIN蛋白表达水平均显著高于对照组（P<0.05）。【结论】在鸡胚原代肝细胞培养基中添加400 μmol·L^-1油酸诱导24 h可建立肝细胞早期脂沉积模型，且48 h时炎症发生，72 h 发生纤维化。

关键词: 油酸, 脂沉积, 鸡, 原代肝细胞

Abstract:

【Background】 Fatty liver syndrome (FLS) is a common nutritional metabolic disease in laying hens during the laying period, which includes a multi-stage pathogenesis process, such as “simple steatosis, steatohepatitis, liver fibrosis, and cirrhosis”, causing substantial economic losses in poultry industry. The construction of fatty liver model is the key to exploring the pathogenesis of FLS, screening nutritional regulation measures and subsequent disease prevention and evaluation. However, there are few studies on poultry hepatocyte steatosis models. 【Objective】 Chicken embryo liver (CEL) cells were used to establish a liver lipid deposition model by oleic acid induction, aiming to explore the induction time required for different stages of FLS.【Method】 CEL at the age of 18 embryo was used and when CEL cells grew to about 80%, and the medium containing 400 μmol·L^-1 oleic acid was replaced for treatment. After 24 h, 48 h and 72 h of culture, the cells were stained with oil red O, and the lipid content was measured. At the same time, the mRNA and protein levels of genes related to lipid metabolism, inflammation and fibrosis were analyzed.【Result】 Compared with the Control group, there were a large number of red lipid droplets in 24 h group, 48 h group and 72 h group, and the red color gradually deepened with the increase of induction time (P<0.05), indicating that fat droplets in cells were increased. Meanwhile, the content of TG and TC in the liver were higher than those in the Control group (P<0.05). RT-PCR analysis revealed a significant upregulation of ACC, FAS, ELOVL6, and SREBP gene expression related to lipid synthesis following 24 h of oleic acid induction (P<0.05), indicating that oleic acid lipid deposited on cells after 24 h of processing. ELISA results showed that the content of IL-6, IL-1β and TNF-α were not significantly changed in 24 h (P>0.05). However, an upward trend was observed in IL-6 level with prolonged induction durations (P=0.052). Notably, following 72 h of oleic acid treatment, the levels of IL-6 and IL-1β were significantly increased in Control group (P<0.05), which indicated that acid treatment after 48 h cells began to inflammation reaction and increased with the extension of induction time. In addition, compared with the Control group, the expression level of IL-6 protein in the 48 h group was significantly increased, which was consistent with the ELISA results, while the protein expression levels of COL1A1 and FIBRONECTIN were increased (P<0.05). On the contrary, the protein expression levels of IL-6, COL1A1 and FIBRONECTIN were significantly higher than those in the Control group at 72 h (P<0.05). 【Conclusion】 The early lipid deposition model of hepatocytes could be established by 400 μmol·L^-1 oleic acid induction for 24 h. In addition, inflammation and fibrosis would occurr after 48 h and 72 h induction, respectively.

Key words: oleic acid, lipidosis, chicken, primary hepatocyte

王超慧, 张利敏, 孙喜, 李思静, 杨小军, 刘艳利. 油酸诱导鸡胚原代肝细胞脂沉积模型的构建[J]. 中国农业科学, 2024, 57(23): 4806-4814.

WANG ChaoHui, ZHANG LiMin, SUN Xi, LI SiJing, YANG XiaoJun, LIU YanLi. The Model Establishment of Lipid Deposition in Primary Chicken Embryo Liver Cells Induced by Oleic Acid[J]. Scientia Agricultura Sinica, 2024, 57(23): 4806-4814.

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基因 Gene	登录号 Accession number	引物序列 Primer sequences (5′ to 3′)	产物大小 Product size (bp)
β-actin	L_08165	F:ATTGTCCACCGCAAATGCTTC R:AAATAAAGCCATGCCAATCTCGTC	113
ACC	XM_046929960	F:GCTTCCCATTTGCCGTCCTA R:GCCATTCTCACCACCTGATTACTG	185
FAS	NM_205155	F:TTTGGTGGTTCGAGGTGGTA R:CAAAGGTTGTATTTCGGGAGC	212
ELOVL6	XM_046916529	F:GGTGGTCGGCACCTAATGAA R:TCTGGTCACACACTGACTGC	169
SREBP	XM_046927256	F: GCCCTCTGTGCCTTTGTCTTC R: ACTCAGCCATGATGCTTCTTC	130