黄花蒿醇提物对奶牛乳腺细胞中共轭亚油酸 合成相关酶基因表达的作用

doi:10.3864/j.issn.0578-1752.2019.18.018

摘要/Abstract

摘要： 目的通过奶牛乳腺上皮细胞体外培养试验,研究黄花蒿乙醇提取物对乳腺上皮细胞中与共轭亚油酸（CLA）合成相关酶的基因SCD表达的影响,探讨其对乳脂肪合成相关酶ACACA和FASN基因以及与脂肪转运相关酶LPL基因表达的影响,旨在从乳腺层次探明黄花蒿乙醇提取物对乳中CLA合成的部分作用机制。方法将采自于健康泌乳中期荷斯坦奶牛的乳腺组织用保温盒带回实验室,在无菌条件选取腺泡较多的深层组织,采用胶原酶消化法获得乳腺上皮细胞,放于37℃,CO₂浓度为5%的培养箱中进行原代培养。试验所用的细胞是经过复苏的第二代细胞,待细胞复苏后,以3×10 ⁴个/mL的密度接种细胞到24孔板中,分别置于37 ℃的5% CO₂培养箱中培养,采用台盼蓝计数法每天进行一次计数,设3个重复,连续7 d,未计数组每2天换液一次,绘制细胞生长曲线。另外,待细胞生长至对数增殖期,更换新鲜培养液,随机分为4组,即培养液中黄花蒿提取物的浓度为0、3.0、6.0、12.0 mg·L ^-1 ,提取物作用时间均为48 h,检测不同浓度黄花蒿提取物对与脂肪酸合成相关酶SCD、ACC、FAS、LPL 基因表达量的影响。每个处理3个重复。结果使用倒置显微镜观察,乳腺上皮细胞形态呈铺路石样;在3×10 ⁴个/mL的接种密度下,细胞生长曲线呈S型,在1—2d乳腺上皮细胞为潜伏期,3—6d为指数增长期,之后进入平台期,符合一般细胞生长曲线规律,说明培养的乳腺上皮细胞具有正常的增殖能力,可以用于后续的研究;与对照组相比,添加黄花蒿乙醇提取物有增加SCD酶基因表达量的趋势,其中3mg·L ^-1组显著增加了SCD酶的基因表达量(P<0.05),而6 mg·L ^-1组和12mg·L ^-1组虽然增加了SCD酶的基因表达量,但与对照组相比差异不显著(P>0.05);添加黄花蒿乙醇提取物有增加乳腺上皮细胞中ACACA基因表达量的趋势,但与对照组相比差异不显著(P>0.05),且随着添加剂量的增加,有下降趋势;添加黄花蒿乙醇提取物可以增加乳腺上皮细胞中FASN基因表达量,呈剂量依赖型增加,且12mg·L ^-1组可以显著增加乳腺上皮细胞中FASN基因表达量(P<0.05);添加黄花蒿乙醇提取物有降低乳腺上皮细胞中LPL基因表达量的趋势,呈剂量依赖型降低,且6 mg·L ^-1组和12mg·L ^-1组显著降低了乳腺上皮细胞中LPL基因表达量(P<0.05)。结论黄花蒿乙醇提取物可以增加奶牛乳腺上皮细胞中SCD、ACACA和FASN酶基因的表达,有利于调控CLA和乳脂肪的生成。

关键词: 黄花蒿乙醇提取物, 奶牛乳腺上皮细胞, SCD, ACACA, FASN, LPL

Abstract:

【Objective】The effects of Artemisia annua extracts (AAE) on gene SCD, ACACA, FASN and LPL related to CLA and milk fat synthesis in the bovine mammary epithelial cells(BMECs) culture in vitro were studied in this paper. The purpose of this study was to explore the effects of AAE on the part mechanism of effecting CLA synthesis from the mammary.【Method】 We isolated BMEC by using tissue fragments from the mammary gland of a healthy Holstein cows in Mid lactation. The cells used in the experiment were the second generation ones which have been resuscitated. The cells were inoculated into 24-well plates at a density of 3×10 ⁴ cells/mL and cultured at 37℃ in a 5% CO₂ atmosphere. The cells were counted by trypan blue counting method once a day. The experiment lasted for 7 d to draw the growth curve of the cells with three replicates, and the uncounted groups changed liquid every 2 d. When the cells grew to logarithmic proliferation stage, fresh culture medium was replaced and randomly divided into four groups, and the concentration of AAE in culture medium was 0, 3.0, 6.0, and 12.0 mg·L ^-1 individually for 48 h. The effects of different concentration of AAE on the gene expression of SCD, ACC, FAS and LPL related to fatty acid synthesis were detected. Each treatment was repeated for three times. 【Result】 The results showed as follows: the morphology and the growth curve for the BMECS looked like paving appearance and S-shaped respectively at 3×10 ⁴ cfu/mL. The latent period and the exponential growth period for the BMECS was 1-2 d and 3-6 d, respectively, and then in the plateau stage, which were accorded with the general cells growth curve rule and showed that the cultured mammary epithelial cells had normal proliferation ability as used in the subsequent researches. Compared with the control group, there was the trend that AAE supplementation increased the expression of SCD enzyme gene, 3 mg·L ^-1 group significantly increased the expression of SCD gene (P<0.05), while the 6 mg·L ^-1 group and the 12 mg·L ^-1 group increased the expression of SCD gene, but there was no significant difference compared with the control group (P>0.05). The addition of AAE increased the expression of ACACA gene in mammary epithelial cells (P>0.05). The addition of AAE increased the expression of FASN gene with a dose-dependent manner, the 12mg·L ^-1 group increased the expression of FASN gene significantly (P<0.05). There was the trend that AAE supplementation decreased the expression of LPL enzyme gene with a dose-dependent manner, and the 6 mg·L ^-1 group and the 12 mg·L ^-1 group decreased the expression of LPL gene significantly (P<0.05). 【Conclusion】 It was concluded that AAE supplementation could increase the expression of SCD, ACACA and FASN genes in BMECS, which was beneficial to CLA synthesis.

Key words: Artemisia annua extracts, BMECS, SCD, ACACA, FASN, LPL

王丽芳,张兴夫. 黄花蒿醇提物对奶牛乳腺细胞中共轭亚油酸合成相关酶基因表达的作用[J]. 中国农业科学, 2019, 52(18): 3271-3278.

WANG LiFang,ZHANG XingFu. Effects of Artemisia annua Extracts on CLA Synthesis and Mechanism[J]. Scientia Agricultura Sinica, 2019, 52(18): 3271-3278.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2019.18.018

https://www.chinaagrisci.com/CN/Y2019/V52/I18/3271

图/表 4

表1

图1

图2

图3

参考文献 35

[1]	SCHMID A, COLLOMB M, SIEBER R, BEE G . Conjugated linoleic acid in meat and meat products: A review. Meat Science, 2006,73:29-41.
[2]	BAUMAN D E, PERFIELD J W I, HARVATINE K J, BAUMGARD L H. Regulation of fat synthesis by conjugated linoleic acid: lactation and the ruminant model. The Journal of Nutrition, 2008,138:403-409.
[3]	BENJAMIN S, SPENER F . Conjugated linoleic acids as functional food: An insight into their health benefits. Nutrition & Metabolism, 2009, 6:36.
[4]	MORAES M L, RIBEIRO A M L, KESSLER A M, LEDUR V S, FISCHER M M, BOCKOR L, GAVA D. Effect of CLA on performance and immune response of weanling piglets. Journal of Animal Science, 2012,90(8):2590-2598.
[5]	OLIVEIRA D E, GAMA M A S, FEMANDES D, TEDESCHI L O, BAUMAN D E. An unprotected conjugated linoleic acid supplement decreases milk production and secretion of milk components in grazing dairy ewes. Journal of Dairy Science, 2012,95:1437-1446.
[6]	DU R, ZHONG T, ZHANG W Q, SONG P, SONG W D, ZHAO Y, WANG C, TANG Y Q, ZHANG X, ZHANG Q . Antitumor effect of iRGD-modified liposomes containing conjugated linoleic acid- paclitaxel (CLA-PTX) on B16-F10 melanoma. International Journal of Nanomedicine, 2014, 9:3091-3105.
[7]	ENGELKE S W, DAS G, DERNO M, TUCHSCHERER A, WIMMERS K, RYCHLIK M, KIENBERGER H, BERG W, KUHLA B, METGES C C . Methane prediction based on individual or groups of milk fatty acids for dairy cows fed rations with or without linseed. Journal of Dairy Science, 2019,102(2) : 1-15.
[8]	BAYAT A R, TAPIO I, VILKKI J, SHINGFIELD K J, LESKINEN H . Plant oil supplements reduce methane emissions and improve milk fatty acid composition in dairy cows fed grass silage-based diets without affecting milk yield. Journal of Dairy Science, 2018,101(2) : 1136-1151.
[9]	王丽芳 . 奶牛日粮中添加整粒胡麻籽和棉籽对牛奶品质的影响[D]. 呼和浩特: 内蒙古农业大学, 2005.
	WANG L F . Effect of linseed and cottonseed on milk quality in lactating dairy cows[D]. Hohhot: Inner Mongolia Agricultural University, 2005. (in Chinese)
[10]	斯琴毕力格, 王丽芳, 丁赫, 刘旺景, 连海飞, 敖长金 . 饲粮中添加黄花蒿乙醇提取物对奶牛产奶性能及乳脂中共轭亚油酸含量的影响. 动物营养学报, 2017,29(11):4137-4142.
	SI Q B L G, WANG L F, DING H, LIU W J, LIAN H F, AO C J . Effects of dietary Artemisia annua extracts on milk performance and conjugated linoleic acid content in milk fat of lactating cows. Chinese Journal of Animal Nutrition, 2017,29(11):4137-4142. (in Chinese)
[11]	王丽芳 . 添加菊科植物黄花蒿提取物对奶山羊乳中CLA含量影响及机理研究[D]. 呼和浩特: 内蒙古农业大学, 2010.
	WANG L F . The study on effects of compositae Artemisia annua extracts on cla content in goat milk and the relative mechanism[D]. Hohhot: Inner Mongolia Agricultural University, 2010. (in Chinese)
[12]	王丽芳, 卢德勋, 高民 . 添加菊科植物黄花蒿乙醇提取物对奶牛乳腺上皮细胞脂肪酸组成的影响. 饲料工业, 2010,31(19):36-38.
	WANG L F, LU D X, GAO M . The effects of compositae Artemisia annua extracts on fatty acid composition of mammary epithelial cells in dairy cows. Feed Industry, 2010,31(19):36-38. (in Chinese)
[13]	COLLOMB M, BUTIKOFER U, SIEBER R, JEANGROS B, BOSSET J O . Correlation between fatty acids in cows’ milk fat produced in the Lowlands, Mountains and Highlands of Switzerland and botanical composition of the fodder. International Dairy Journal, 2002,12(8):661-666.
[14]	CABIDDU A, ADDIS M, PINNA G, SPADA S, FIORI M, SITZIA M, PIRISI A, PIREDDA G, MOLLE G . The inclusion of a daisy plant (Chrysanthemum coronarium) in dairy sheep diet. 1: Effect on milk and cheese fatty acid composition with particular reference to C18: 2 cis-9, trans-11. Livestock Science, 2006,101(1):57-67.
[15]	WALLACE R J . Biohydrogenation of fatty acids in the rumen//The 6th Joint Symposium of China-Korea-Japan on Rumen Metabolism and Physiology. 2007,4.
[16]	王丽芳, 斯琴毕力格, 敖长金. 黄花蒿提取物对奶牛瘤胃发酵指标的影响. 中国农业科学, 2018,51(23):4548-4555. doi: 10.3864/j.issn.0578-1752.2018.23.013
	WANG L F, SI Q B L G, AO C J. The Effects of Artemisia annua extracts on the rumen fermentation in dairy cows. Scientia Agricultura Sinica, 2018,51(23):4548-4555. (in Chinese) doi: 10.3864/j.issn.0578-1752.2018.23.013
[17]	孙攀峰 . 粗饲料组合对瘤胃发酵及乳脂肪酸组成的影响及其机理研究[D]. 杭州: 浙江大学, 2007.
	SUN P F . Effect of Forage Combination on Ruminal Fermentation and Milk Fatty Acids Composition[D]. Hangzhou: Zhejiang University, 2007. (in Chinese)
[18]	张兴夫 . 不同日粮模式对泌乳奶牛乳腺乳蛋白合成影响的研究[D]. 呼和浩特: 内蒙古农业大学, 2013.
	ZHANG X F . Study on effects of different diets on milk protein synthesis in the mammary gland of dairy cows[D]. Hohhot: Inner Mongolia Agricultural University, 2013. (in Chinese)
[19]	塔娜, 李红磊, 侯先志, 考桂兰, 高民, 李大彪 . 乙酸钠和β-羟丁酸钠对奶牛乳腺上皮细胞乳脂和乳蛋白合成相关基因表达的影响. 动物营养学报, 2014,26(6):1527-1534. doi: 10.3969/j.issn.1006-267x.2014.06.013
	TA N, LI H L, HOU X Z, KAO G L, GAO M, LI D B . Effects of sodium acetate and sodium β-hydroxybutyrate on expressions of genes involved in milk fat and protein synthesis in bovine mammary epithelial cells. Chinese Journal of Animal Nutrition, 2014,26(6):1527-1534. (in Chinese) doi: 10.3969/j.issn.1006-267x.2014.06.013
[20]	李大彪, 李红磊, 邢媛媛, 于永强, 王卫云, 陈玲, 李平 . 亚油酸对奶牛乳腺上皮细胞乳脂肪和乳蛋白合成相关基因表达的影响. 中国细胞生物学报, 2016,38(3):257-264.
	LI D B, LI H L, XING Y Y, YU Y Q, WANG W Y, CHEN L, LI P . Effect of linoleic acid on the expression of genes associated with milk fat and milk protein synthesis of bovine mammary epithelial cells. Chinese Journal of Cell Biology, 2016,38(3):: 257-264.
[21]	王治国 . 奶牛乳腺上皮细胞的体外培养及应用[D]. 北京:中国农业科学院, 2007.
	WANG Z G . In vitro culture and application of bovine mammary epithelial cell[D]. Beijing: Chinese Academy of Agricultural Sciences, 2007. (in Chinese)
[22]	YAO D W, LUO J, HE Q Y, LI J, WANG H, SHI H B, XU H F, WANG M, LOOR J J . Characterization of the liver X receptor- dependent regulatory mechanism of goat stearoyl-coenzyme A desaturase 1 gene by linoleic acid. Journal of Dairy Science, 2016,99(5):3945-3957.
[23]	BAUMGARD L H, MATITASHVILI E, CORL B, DWYER D, BAUMAN D . Trans-10, cis-12 conjugated linoleic acid decreases lipogenic rates and expression of genes involved in milk lipid synthesis in dairy cows. Journal of Dairy Science, 2002,85(9):2155-2163.
[24]	GERVAIS R, MCFADDEN J W, LENGI A J, CORL B A, CHOUINARD P Y . Effects of intravenous infusion of trans-10, cis-12 18:2 on mammary lipid metabolism in lactating dairy cows. Journal of Dairy Science, 2009,92(10) : 5167-5177.
[25]	DEANDRADE P V, SCHMIDELY P . Effect of duodenal infusion of trans-10, cis-12-CLA on milk performance and milk fatty acid profile in dairy goats fed high or low concentrate diet in combination with rolled canola seed. Reproduction, Nutrition, Development, 2006,46(1):31-48.
[26]	INVERNIZZI G, THERING B J, MCGUIRE M A, SAVOINI G, LOOR J J . Sustained upregulation of stearoyl-CoA desaturase in bovine mammary tissue with contrasting changes in milk fat synthesis and lipogenic gene networks caused by lipid supplements. Functional Integrative Genomics, 2010,10(4):561-575.
[27]	CHAJÈS V, CAMBOT M, MOREAU K, LENOIR G M, JOULIN V . Acetyl-CoA carboxylase alpha is essential to breast cancer cell survival. Cancer Research, 2006,66(10):5287-5294.
[28]	SALAMA A A K, DUQUE M, WANG L, SHAHZAD K, OLIVERA M, LOOR J J . Enhanced supply of methionine or arginine alters mechanistic target of rapamycin signaling proteins, messenger RNA, and microRNA abundance in heat-stressed bovine mammary epithelial cells in vitro. Journal of Dairy Science, 2019,102(3):1-12.
[29]	ZHANG T Y, HUANG J T, TIAN H B, MA Y, CHEN Z, WANG J J, SHI H P, LUO J . Trans-10, cis-12 conjugated linoleic acid alters lipid metabolism of goat mammary epithelial cells by regulation of de novo synthesis and the AMPK signaling pathway. Journal of Dairy Science, 2018,101(6):5571-5581.
[30]	苏芮, 刘阳, 闫素梅, 史彬林, 赵艳丽, 石惠宇 . 维生素A对奶牛乳腺上皮细胞乳脂和乳蛋白合成相关基因表达的影响. 动物营养学报, 2018,30(8):3151-3158.
	SU R, LIU Y, YAN S M, SHI B L, ZHAO Y L, SHI H Y . Effects of Vitamin A on gene expressions related to milk fat and protein synthesis in bovine mammary epithelial cells. Chinese Journal of Animal Nutrition, 2018,30(8):3151-3158. (in Chinese)
[31]	BIONAZ M, LOOR J J . Gene networks driving bovine milk fat synthesis during the lactation cycle. BMC Genomics, 2008,9:366-387.
[32]	胡仕良 . 西农萨能奶山羊乳腺上皮细胞LPL基因特征与功能的初步研究[D]. 杨凌: 西北农林科技大学, 2012.
	HU S L . Primary characteristics and function study of LPL in epithelial cell in Xinong Saanen dairy goat[D]. Yangling: Northwest A and F University, 2012. (in Chinese)
[33]	茅慧玲 . 植物源性物质对生长湖羊瘤胃发酵和肉品质的影响及其作用机理的研究[D]. 杭州: 浙江大学, 2010.
	MAO H L . Effects of plants-based materials on growth performance, rumen fermentation and meat quality of growing Hu lambs[D]. Hangzhou: Zhejiang University, 2010. (in Chinese)
[34]	张博綦 . 硒对奶牛乳腺上皮细胞内乳脂肪和乳蛋白合成相关基因表达的影响[D]. 呼和浩特: 内蒙古农业大学, 2017.
	ZHANG B Q . Effects of selenium on gene expression related to milk fat and protein synthesis in bovine mammary epithelial cells[D]. Hohhot: Inner Mongolia Agricultural University, 2017. (in Chinese)
[35]	朱智 . 大蒜油对山羊瘤胃生物氢化、乳脂肪酸组成及乳腺脂肪生成相关基因表达影响的研究[D]. 南京: 南京农业大学, 2011.
	ZHU Z . Effects of garlic oil on ruminal biohydrogenation, milk fatty acid profile and lipogenesis-related gene expression in mammary gland of goats[D]. Nanjing: Nanjing Agricultural University, 2011. (in Chinese)

基因名称 Gene	引物序列 Primer sequence	Gene bank序列号 Gene bank accessions No.	扩增片段 Amplicon size (bp)
内参基因 β-Actin	F. 5′-AACTCCATCATGAAGTGTGACG	NM_173979.3	214
内参基因 β-Actin	R. 5′- GATCCACATCTGCTGGAAGG		214
硬脂酰辅酶A去饱和酶 SCD	F. 5′- TCCTGTTGTTGTGCTTCATCC	AY241933	101
硬脂酰辅酶A去饱和酶 SCD	R. 5′- GGCATAACGGAATAAGGTGGC		101
乙酰辅酶A羧化酶 ACACA	F. 5′- CATCTTGTCCGAAACGTCGAT	AJ132890	101
乙酰辅酶A羧化酶 ACACA	R. 5′- CCCTTCGAACATACACCTCCA		101
脂肪酸合成酶 FASN	F. 5′-ACCTCGTGAAGGCTGTGACTCA	CR552737	92
脂肪酸合成酶 FASN	R. 5′-TGAGTCGAGGCCAAGGTCTGAA		92
脂蛋白酯酶 LPL	F. 5′- ACACAGCTGAGGACACTTGCC	BC118091	101
脂蛋白酯酶 LPL	R. 5′- GCCATGGATCACCACAAAGG		101