鸡原代肝细胞培养及叶酸对脂质代谢相关基因表达的影响

doi:10.3864/j.issn.0578-1752.2017.21.015

Abstract

Abstract: 【Objective】Abdominal fat deposition is a universal phenomenon at present in poultry industry. Different from mammals, 90%-95% of lipid metabolism in poultry occurs in the liver. Previous studies have demonstrated folic acid and IGF2 were involved in animals lipid metabolism. The study was conducted to establish the method for primary culture of chicken hepatocytes and investigate the effects of folic acid on IGF2 and genes expression associated with lipid metabolism in primary chicken hepatocytes, further providing a basis for exploring lipid metabolism of poultry in vitro. 【Method】Chicken hepatocytes were isolated by collagenase digestion combined with purification of chicken lymphocyte separation fluid, and evaluated by PAS staining, respectively. After 36 h culture of hepatocytes, cells were treated with different concentration of folic acid for 12 h. Hepatocytes proliferation and injury was detected by MTT method and LDH activity in culture medium respectively. Cells were collected to get total RNA for gene expression analysis by RT-PCR. Later, correlation analysis was carried out between IGF2 and lipid metabolism related genes expression. Regression analysis was performed between folic acid concentration and genes expression.【Result】The results showed that hepatocytes were isolated with high purity. Folic acid didn’t affect cell proliferation and cellular LDH activity (P＞0.05).When compared with the control group (1 mg·L^-1 folic acid), 10, 15 and 20 mg·L^-1 folic acid significantly reduced IGF2 expression in chicken hepatocytes (P＜0.05), and same phenomenon was observed in FAS and ACC expression. However, folic acid had no effects on CPT-1 and PPARα expression (P＞0.05) . In addition, FAS and ACC expression had positive correlation with IGF2 mRNA level in chicken hepatocytes (P＜0.05). There existed linear and quadratic regression between genes expression and folic acid concentration (P＜0.05). 【Conclusion】 In conclusion, folic acid could reduce gene expression associated with fatty acid synthesis in chicken hepatocytes. In addition, FAS and ACC mRNA level might have positive connection with IGF2 expression. Optimum treatment dose of folic acid was 15 mg·L^-1 in this study.

Key words: folic acid, IGF2, hepatocytes, lipid metabolism, chicken

LIU YanLi, DANG YanNa, DUAN YuLan, YANG XiaoJun. Effect of Folic Acid on Lipid Metabolism Associated Gene Expression in Primarily Cultured Chickens Hepatocytes[J].Scientia Agricultura Sinica, 2017, 50(21): 4205-4211.

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References

[1] LEVEILLE GILBERT A, ROMSOS DALE R, YEH Y Y, O’HEA E K. Lipid biosynthesis in the chick. A consideration of site of synthesis, influence of diet and possible regulatory mechanisms. Poultry Science, 1975, 54(4): 1075-1093.

[2] 闻治国. 胆碱对北京鸭生长发育和脂肪代谢的影响及其调控机制 [D]. 北京: 中国农业大学, 2015.

WEN Z G. The effect of choline on growth performance and lipid metabolism in Pekin ducks and its regulation mechanism [D]. Beijing: China Agricultural University, 2015. (in Chinese)

[3] 喻小琼. 叶酸对鸡脂肪沉积的影响及表观遗传调控机制研究 [D]. 北京: 中国农业科学院, 2014.

YU X Q. Study of the effect of folic acid on fat deposition and the underlying epigenetic mechanisms in chicken [D]. Beijing: Chinese Academy of Agricultural Sciences, 2014. ( in Chinese)

[4] 王美玲. 锰对原代培养肉鸡肝细胞脂肪合成关键酶活性及其基因表达的影响 [D]. 北京: 中国农业科学院, 2011.

WANG M L. Effect of manganese on activities and gene expression of key enzymes in fat synthesis in primary cultured hepatocytes of broilers [D]. Beijing: Chinese Academy of Agricultural Sciences, 2011. ( in Chinese)

[5] WANG S P, YIN Y L, QIAN Y, LI L L, LI F N, TAN B E, TANG X S, HUANG R L. Effects of folic acid on the performance of suckling piglets and sows during lactation. Journal of the Science of Food and Agriculture, 2011, 91(13): 2371-2377.

[6] 姚英, 陈代文, 刘静波, 毛湘冰, 毛倩, 余冰. 叶酸对超早期断奶宫内发育迟缓仔猪肝脏结构和细胞凋亡相关基因表达的影响. 动物营养学报, 2012, 24(2): 271-279.

YAO Y, CHEN D W, LIU J B, MAO X B, MAO Q, YU B. Folic acid in intrauterine growth retarded early weaner piglets: effects on hepatic structure and apoptosis-related gene expression. Chinese Journal of Animal Nutrition, 2012, 24(2): 271-279. ( in Chinese)

[7] BURDGE G C, LILLYCROP K A, JACKSON A A, GLUCKMAN P D, HANSON M A. The nature of the growth pattern and of the metabolic response to fasting in the rat are dependent upon the dietary protein and folic acid intakes of their pregnant dams and post-weaning fat consumption. British Journal of Nutrition, 2008, 99(3): 540-549.

[8] 葛文霞. 烟酸和不同水平叶酸对肉仔鸡生产性能和血清理化指标影响的研究 [D]. 石河子: 石河子大学, 2006.

GE W X. Effect of nicotinic acid and different levels of folic acid on growth performance and physiological and biochemical serum indexes in broilers [D]. Shihezi: Shihezi Unixersity. 2006. ( in Chinese)

[9] ZHANG J, ZHANG F, DIDELOT X, BRUCE K D, CAGAMPANG F R, VATISH M, BYRNE C D. Maternal high fat diet during pregnancy and lactation alters hepatic expression of insulin like growth factor-2 and key microRNAs in the adult offspring. BMC Genomics, 2009, 10: 478.

[10] SPENCER G S, DECUYPERE E, BUYSE J, ZEMAN M. Effect of recombinant human insulin-like growth factor-II on weight gain and body composition of broiler chickens. Poultry Science, 1996, 75(3): 388-392.

[11] ZHI H L, HUI L, QI G W, ZHAO J, WANG Y. The study on correlation analysis of single nucleotide polymorphism of IGF2 gene and body fatness traits in chicken. Agricultural Sciences in China, 2004, 3(10): 789-794.

[12] 刘艳利, 申静, 支丽慧, 李世召, 姚军虎, 杨小军. 叶酸调控鸡脾和胸腺 IGF2 表达的表观遗传机制探究. 畜牧兽医学报, 2016, 47(2): 296-304.

LIU Y L, SHEN J, ZHI L H, LI S Z, YAO J H, YANG X J. The study on epigenetic mechanism of IGF2 expression in spleen and thymus regulated by folic acid in broilers. Acta Veterinaria Et Zootechnica Sinica, 2016, 47(2): 296-304.( in Chinese)

[13] 陈黎龙, 江青艳, 朱晓彤, 束刚, 高淑静, 高萍, 张永亮. 成年鸡肝细胞的分离与原代培养. 江西农业大学学报, 2008, 30(3): 385-389.

CHEN L L, JIANG Q Y, ZHU X T, SHU G, GAO S J, GAO P, ZHANG Y L. Separation and primary culture of adult chicken hepatocytes. Journal of Jiangxi Agriculture university, 2008, 30(3): 385-389. ( in Chinese)

[14] 刘腾飞, 田静, 李会敏, 余祖功. 半原位灌流法分离成年鸡肝细胞及原代培养. 畜牧与兽医, 2015(6): 115-118.

LIU T F, TIAN J, LI H M, YU Z G. Primary culture and isolation hepatocytes in chicken by half perfusion method. Animal Husbandry and Veterinary Medicine, 2015(6): 115-118. ( in Chinese)

[15] 江青艳, 傅伟龙, 高淑静, 吴荣辉. 鸡离体肝细胞几种分离方法的比较. 华南农业大学学报, 2001, 22(1): 66-69.

JIANG Q Y, FU W L, GAO S J, WU R H. Comparison of several methods for isolating chicken hepatocytes. Journal of South China Agricultural University, 2001, 22(1): 66-69. ( in Chinese)

[16] LEGRAND P, CATHELINE D, LE B E, LEMARCHAL P. Effect of insulin on triacylglycerol synthesis and secretion by chicken hepatocytes in primary culture. The International Journal of Biochemistry & Cell Biology, 1996, 28(4): 431-440.

[17] LIVAK K J, SCHMITTGEN T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2^-ΔΔCT method. Methods, 2001, 25(4): 402-408.

[18] 刘腾飞, 田静, 耿智霞, 李会敏, 余祖功. 抗菌药物对 LPS 诱导鸡肝细胞损伤的影响及复方甘草酸单胺的修复效应. 畜牧兽医学报, 2015, 46(2): 309-316.

LIU T F, TIAN J, GENG Z X, LI H M, YU Z G. Effect of antibiotic on LPS induced licer cell injury and hepatoprotective acitivity of compound ammonium glycyrrhizin. Acta Veterinaria Et Zootechnica Sinica, 2015, 46(2): 309-316. ( in Chinese)

[19] SEGLEN P O. Preparation of isolated rat liver cells. Methods in Cell Biology, 1976, 13: 29-83.

[20] LI J, LEGHARI I H, HE B, MI Y, ZHANG C. Estrogen stimulates expression of chicken hepatic vitellogenin II and very low-density apolipoprotein II through ER-alpha. Theriogenology, 2014, 82(3): 517-524.

[21] PUVIANI A C, OTTOLENGHI C, TASSINARI B, PAZZI P, MORSIANI E. An update on high-yield hepatocyte isolation methods and on the potential clinical use of isolated liver cells. Comparative

Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 1998, 121(2): 99-109.

[22] YAMANAKA N, KITANI H, MIKAMI O, NAKAJIMA Y, MIURA K. Serum-free culture of adult chicken hepatocytes; morphological and biochemical characterization. Research in Veterinary Science, 1997, 62 (3): 233-237.

[23] 蒋志惠. 硒蛋白W对鸡肝脏细胞凋亡影响的研究[D]. 哈尔滨: 东北农业大学, 2013.

JIANG Z H. The effect of selW on apoptosis in liver cells of chicken [D]. Harbin: Northeast Agricultural University. 2013. ( in Chinese)

[24] CHENG Y B, WANG Y J, ZHANGS C, LIU J, CHEN Z, LI J J. Response of porcine hepatocytes in primary culture to plasma from severe viral hepatitis patients. World Journal of Gastroenterology, 2005, 11(48): 7585.

[25] ROGLER C E, YANG D Y, ROSSETTI L, DONOHOE J, ALT E, CHANG C J, ROSENFELD R, NEELY K, HINTZ R. Altered body composition and increased frequency of diverse malignancies in insulin-like growth factor-II transgenic mice. Journal of Biological Chemistry, 1994, 269(19): 13779-13784.

[26] JONES B K, LEVORSE J, TILGHMAN S M. Deletion of a nuclease-sensitive region between the Igf2 and H19 genes leads to Igf2 misregulation and increased adiposity. Human Molecular Genetics, 2001, 10(8): 807-814.

[27] WANG H B, LI H, WANG Q G, ZHANG X Y, WANG S Z, WANG Y X, WANG X P. Profiling of chicken adipose tissue gene expression by genome array. BMC Genomics, 2007, 8: 193.

[28] 喻小琼, 刘冉冉, 赵桂苹, 郑麦青, 李鹏, 文杰. 叶酸对种鸡子代胚胎期脂代谢基因表达及甲基化的影响. 动物营养学报, 2014, 26(7): 1796-1806.

YU X Q, LIU R R, ZHAO G P, ZHENG M Q, LI P, WEN J. Effects of maternal folate supplementation for breeding chickens on lipid metabolism-related gene expression and methylation in offspring during embryonic development. Chinese Journal of Animal Nutrition, 2014, 26(7): 1796-1806. ( in Chinese)

[29] YU X Q, LIU R R, ZHAO G P, ZHENG M Q, CHEN J, WEN J. Folate supplementation modifies CCAAT/enhancer-binding protein α methylation to mediate differentiation of preadipocytes in chickens. Poultry Science, 2014, 93 (10): 2596-2603.

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