Abstract：Stearoyl-CoA desaturase 1 (SCD1) is a rate-limiting enzyme for the synthesis of monounsaturated fatty acids in hepatocytes, and plays an important regulation role in the composition of fatty acids in the body. In order to explore the genetic regulation mechanism of SCD1 gene on the fat deposition of poultry, 21 days embryo duck (Anas platyrhynchos) hepatocytes were extracted by the method of type Ⅳ collagenaseand. The pEGFP-N3-SCD1 eukaryotic over-expression vector was constructed and transferred into the hepatocytes. The over-expression of SCD1 gene in duck hepatocytes was measured by HIS labeling assay kit to understand the effect of SCD1 on lipid metabolism. The experimental results showed that initial separation of hepatocytes was good, and the cells were basically covered with culture plates until 96 h, The recombinant plasmid pEGFP-N3-SCD1 and green fluorescence could over- expression stably in the primary hepatocytes of the ducks. The correlation between over-expression and lipid index showed that the expression of SCD1 was positively correlated with total cholesterol (TC) and low density lipoprotein (LDL) (P＜0.01), The content of high density lipoprotein (HDL) was significantly negative correlation (P＜0.01) but triglyceride (TG) (P＞0.05). The results showed that the recombinant plasmid pEGFP-N3-SCD1 could be successfully isolated and purified from the liver extraction by using type Ⅳ collagenase. Over-expression of SCD1 gene had a regulatory function for lipid metabolism of duck primary hepatocytes and it is useful for the future study about the genetic regulation mechanism of SCD1 on duck fat deposition.
蔡德丰, 范建高, 马东礼,等. 2014. 硬脂酰辅酶A去饱和酶-1过表达影响高脂诱导的肝细胞 凋亡[J]. 中华肝脏病杂志, 22(1). (Cai D F, Fan J G, Ma D L, et al. 2014. SCD1 over-expression inhibits palmitic acid-induced apoptosis of rat BRL hepatocytes[J]. Chin J of Hepatol, 22(1). ) 黄翼然. 2012. 肾透明细胞癌中硬脂酰辅酶A去饱和酶1蛋白功能及其表达调控机制的研究 [D].博士学位论文，上海交通大学，导师：张宇坚，pp.5-6. ( Huang Y R. 2012. The Expression and Regulation of Stearoyl-COA Desaturases1 in Clear Cell Renal Cell Carcinoma[D]. Ph.D., Shanghai Jiaotong University, Suppervisor: Zhang Y J, pp.5-6.) 刘三凤, 任军, 郭源梅,等.2008. 鸡SCD和Sirt1基因的SNP搜寻及其遗传多样性分析[J]. 畜 牧兽医学报, 39(3):278-285. (Liu S F, Ren J, Guo Y M, et al. 2008. Identification of Single Nucleotide Polymorphisms of SCD and Evaluation of Their Genetic Diversities in Chickens[J]. Acta Veterinaria et Zootechnica Sinica, 39(3):278-285.) 李卫华, 杨佳欣.2014. 硬脂酰辅酶A去饱和酶1与恶性肿瘤的研究进展[J]. 中国肿瘤临床41(17):1131-1134. (Li W H, Yang J X. 2014. Development in the Study of the Correlation between stearoyl-coenzyme A Desaturase 1 and Cancer Progression[J]. Chin J Oncol,41(17):1131-1134.) 李万贵, 张依裕, 王单单,等.2016. 鸭SCD1基因对PC3细胞脂质性状的效应分析[J]. 基因组学与应用生物学, 35(9):2355-2360.( Li W G, Zhang Y Y, Wang D D, et al. 2016. Effect analysis of duck SCD1 gene on lipid traits of PC3 cells[J]. Genomics and annlied aioloev, 35(9): 2355-2360.) 徐雅玲, 黄巨恩, 梁菁,等.2016. 改构型酸性成纤维细胞生长因子对紫杉醇诱导体外大鼠肝细胞损伤保护作用的体外实验研究[J]. 暨南大学学报(自然科学与医学版), 37(1):44-48. ((Xu Y L, Huang J E, Liang J, et al .2016. Protective effects of MaFGF on the injury of hepatocytes induced by taxol in vitro[J]. Journal of jinan university (Natural science&medicine edition), 37(1): 44-48.) 余凯凡. 2010. 不同品种猪肌肉脂肪酸组成的比较及相关功能基因的筛选与鉴定[D]. 硕士学位论文, 华南农业大学, 导师: 江青艳, pp.19-20. (Yu K F. 2010. Screening and identification of related functions and comparison of porcine muscle fatty acid composition of different varieties of gene[D]. Thesis for M.S., South china agricultural university,Suppervisor: Jiang Q Y, pp.19-20. ) 郑东旖, 樊超楠, 刘兆秋, 等. 2010. 学龄前肥胖儿童硬脂酰辅酶A去饱和酶的变化[J]. 中妇 幼保健, 25(16):2225-2227. (Zheng D Y, Fan C N, Liu Z Q, et al. 2010. Changes of stearoyl coenzylme A desaturase in preschool children with obesity[J].Maternal and child health care of china, 25(16): 2225-2227.) 张静静, 宋玉芹, 李桢, 等. 2013. 中国西门塔尔牛SCD1基因多态性与肉质性状的相关分析 [J]. 华北农学报, 28(1): 54-57.(Zhang J J, Song Y Q, Li Z, et al. 2013. Polymorphisms of SCD1 gene and its association with meat quality traits in chinese simmental[J]. Acta Agriculturae Boreali-singca,28(1): 54-57.) 朱才业, 韦光辉, 李伟,等.2013. 徐淮山羊SCD1基因的克隆和亚细胞定位研究及转基因小鼠 的制备[J]. 中国农业科学, 46(17):3695-3703.（Zhu C Y, Wei G H, Li W, et al.2013. Cloning of xuhuai goat SCD1 gene，subcellular localization and the preparation of transgenic mice[J]. Scientia agricultura sinica,46(17):3695-3703. 张宜辉, 张蕊, 张军,等.2014. 鹅SCD1基因真核表达载体构建及在肝细胞中的表达[J]. 上海 交通大学学报:农业科学版, 32(2):5-9.(Zhang Y H,Zhang R,Zhang J,et al.2014. Construction and expression of goose SCD 1 gene in primary hepatocyte[J]. Journal of shanghai jiaotong university : Agricul turil science, 32(2):5-9.) Angelucci C, Maulucci G, Colabianchi A, et al. 2015. Stearoyl-CoA desaturase 1 and paracrine diffusible signals have a major role in the promotion of breast cancer cell migration induced by cancer-associated fibroblasts[J]. British Journal of Cancer, 112(10):1675-1686. Cleghorn G J. 2006. Liver metabolism[C]//1st Int Symp & 4th Int Course on Metabolism & Clinical Nutrition.PP. 12-12. Donato M T, Castell J V, Gomez-Lechon M J. 1999. Characterization of drug metabolizing activities in pig hepatocytes for use in bioartificial liver devices: comparison with other hepatic cellular models[J]. Journal of Hepatology, 31(3): 542-549. Dodgson S J, Forster R E, Storey B T.1984. The role of carbonic anhydrase in hepatocyte metabolism[J]. Annals of the New York Academy of Sciences, 429(1): 516-524. Edge S B, Hoeg J M, Triche T, et al.1986. Cultured human hepatocytes. Evidence for metabolism of low density lipoproteins by a pathway independent of the classical low density lipoprotein receptor[J]. Journal of Biological Chemistry, 261(8): 3800-3806. Foretz M, Pacot C, Dugail I, et al.1999. ADD1/SREBP-1c is required in the activation of hepatic lipogenic gene expression by glucose[J]. Molecular & Cellular Biology, 19(5): 3760. Huang G M, Jiang Q H, Cai C, et al.2015. SCD1 negatively regulates autophagy-induced cell death in human hepatocellular carcinoma through inactivation of the AMPK signaling pathway[J]. Cancer Letters, 358(2): 180-190. Jeffcoat R, James A T.1978. The control of stearoyl-CoA desaturase by dietary linoleic acid[J]. Febs Letters, 85(1): 114-122. Liu M L, Mars W M, Zarnegar R. 1994. Collagenase pretreatment and the mitogenic effects of hepatocyte growth factor and transforming growth factor-α in adult rat liver[J]. European Journal of Neuroscience,19(6): 1521-1527. Maria, Jaureguiberry, Alejandra, et al. 2014. Role of plasma membrane lipid composition on cellular homeostasis: learning from cell line models expressing fatty acid desaturases[J]. Acta Biochimica Et Biophysica Sinica, 46(4): 273-282. Matsusue K, Aibara D, Hayafuchi R, et al. 2014. Hepatic PPARγ and LXRα independently regulate lipid accumulation in the livers of genetically obese mice[J]. Febs Letters, 588(14): 2277-81. Miyazaki M, Kim Y, Ntambi J. 2001. A lipogenic diet in mice with a disruption of the stearoyl-CoA desaturase 1 gene reveals a stringent requirement of endogenous monounsaturated fatty acids for triglyceride synthesis[J]. Journal of Lipid Research, 42(7): 1018-1024. Paton C M, Ntambi J M.2009. Biochemical and physiological function of stearoyl-CoA desaturase[J]. American Journal of Physiology, 297(1): E28-37. Pichard L, Raulet E, Fabre G, et al.1986. Human hepatocyte culture[J]. Methods in Molecular Biology, 8(1): 33-50. Ralston J C, Metherel A H, Stark K D, et al. 2016. SCD1 mediates the influence of exogenous saturated and monounsaturated fatty acids in adipocytes: Effects on cellular stress, inflammatory markers and fatty acid elongation[J]. Journal of Nutritional Biochemistry, 27(1): 241-8.
