玉米赤霉烯酮对奶牛乳腺上皮细胞生长和乳脂合成相关基因表达的影响

doi:10.7668/hbnxb.20194501

华北农学报 ›› 2024, Vol. 39 ›› Issue (1): 201-210. doi: 10.7668/hbnxb.20194501

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玉米赤霉烯酮对奶牛乳腺上皮细胞生长和乳脂合成相关基因表达的影响

马梓峰¹, 李巧¹, 徐红梅¹, 李悦悦¹, 殷实¹^,²^,³, 何翃闳¹^,²^,³, 熊燕¹^,²^,³, 兰道亮¹^,²^,³, 李键¹^,²^,³, 熊显荣¹^,²^,³, 付伟¹^,²^,³

¹ 西南民族大学畜牧兽医学院,四川成都 610041
² 西南民族大学青藏高原动物遗传资源保护与利用教育部重点实验室,四川成都 610041
³ 西南民族大学动物科学国家民委重点实验室,四川成都 610041

收稿日期:2023-11-12 出版日期:2024-02-28
通讯作者:
付伟(1989—),男,四川资阳人,讲师,博士,主要从事动物繁育与胚胎工程研究。
作者简介:
马梓峰(1997—),男,黑龙江牡丹江人,在读硕士,主要从事动物遗传育种与繁殖研究。
基金资助:
四川省自然科学基金项目(22NSFSC2929); 国家重点研发计划项目(2021YFD1600200); 西南民族大学引进人才科研启动金资助项目(RQD2021073)

The Effect of Zearalenone on the Growth of Bovine Mammary Epithelial Cells and the Expression of Genes Related to Milk Fat Synthesis

MA Zifeng¹, LI Qiao¹, XU Hongmei¹, LI Yueyue¹, YIN Shi¹^,²^,³, HE Honghong¹^,²^,³, XIONG Yan¹^,²^,³, LAN Daoliang¹^,²^,³, LI Jian¹^,²^,³, XIONG Xianrong¹^,²^,³, FU Wei¹^,²^,³

¹ College of Animal & Veterinary Sciences,Southwest Minzu University,Chengdu 610041,China
² Key Laboratory of Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Utilization of Ministry of Education,Southwest Minzu University,Chengdu 610041,China
³ Key Laboratory of Animal Science of National Ethnic Affairs Commission,Southwest Minzu University,Chengdu 610041,China

Received:2023-11-12 Published:2024-02-28

摘要/Abstract

摘要：

旨在探索不同浓度玉米赤霉烯酮(ZEN)对奶牛乳腺上皮细胞(MAC-T)生长和乳脂合成相关基因表达的影响。首先,用不同剂量ZEN处理MAC-T细胞36 h,血细胞计数板统计细胞数量,染色并分析细胞凋亡与坏死情况,筛选ZEN添加的合适剂量;接着,试剂盒检测ZEN对MAC-T细胞中活性氧(ROS)以及线粒体膜电位的影响;最后,利用实时荧光定量PCR(RT-qPCR)技术,分析ZEN对MAC-T细胞增殖、凋亡、氧化应激和乳脂合成相关基因表达的影响。结果表明,低剂量ZEN(0.01~1.00 μmol/L)有促进MAC-T生长的趋势,而高剂量ZEN(5.00~10.00 μmol/L)显著降低MAC-T细胞数量;0.10 μmol/L ZEN对MAC-T中ROS和线粒体数量无明显影响,但10.00 μmol/L ZEN显著增加了MAC-T中ROS水平,降低了线粒体数量;RT-qPCR结果表明,0.10 μmol/L ZEN显著促进增殖基因(CDK1、CCND2)、抗氧化基因(DHODH、GPX4)和抗凋亡基因(BCL-2)表达,但同时提高了凋亡基因(CAS-3、BAX)表达量;10.00 μmol/L ZEN显著抑制增殖基因(PCNA、CDK1、CCND2)、抗氧化基因(DHODH、GPX4、AIFM2)和抗凋亡基因(BCL-2)表达,但显著促进凋亡基因(CAS-3、BAX)表达;值得注意的是,0.10 μmol/L ZEN显著促进了乳脂合成相关基因(PPARγ、FASN、JAK-2),但10.00 μmol/L ZEN显著抑制了这些基因表达。上述结果提示,不同浓度ZEN对MAC-T细胞的作用存在差异:0.10 μmol/L ZEN可以促进MAC-T细胞生长和乳脂合成相关基因表达的同时,也会诱导凋亡基因表达;10.00 μmol/L ZEN会诱导MAC-T细胞氧化应激、降低线粒体数量,抑制增殖、抗氧化、抗凋亡和乳脂合成相关基因表达,同时促进凋亡基因表达,导致细胞死亡。

关键词: 奶牛, 玉米赤霉烯酮, 乳腺上皮细胞, 乳脂合成, 增殖, 凋亡

Abstract:

This study aims to explore the effects of different concentrations of zearalenone (ZEN) on the growth of bovine mammary alveolar cell-T (MAC-T) and the expression of genes related to milk fat synthesis.Firstly,MAC-T cells were treated with different doses of ZEN for 36 hours,and the number of cells was counted using a hemocytometer.Then,cells were stained and analyzed for apoptosis and necrosis to determine the appropriate dose of ZEN.Next,acommercial detection kit was used to examine the impact of ZEN on reactive oxygen species (ROS) and mitochondria number in MAC-T cells.Finally,using Real-time Quantitative PCR (RT-qPCR) technology,the influence of ZEN on proliferation,apoptosis,oxidative stress,and milk fat synthesis-related gene expression in MAC-T cells was analyzed.The results showed that low doses of ZEN (0.01—1.00 μmol/L) tended to promote MAC-T growth,whereas high doses of ZEN (5.00—10.00 μmol/L) significantly reduced the number of cells,0.10 μmol/L ZEN had no obvious effect on ROS and mitochondria number in MAC-T,but 10.00 μmol/L ZEN notably elevated ROS levels and decreased mitochondria number.RT-qPCR results indicated that 0.10 μmol/L ZEN significantly promoted the expression of proliferation genes (CDK1,CCND2),antioxidant genes (DHODH,GPX4),and anti-apoptotic genes (BCL-2),but also increased the expression of apoptotic genes (CAS-3,BAX).Whereas,10.00 μmol/L ZEN significantly inhibited the expression of proliferation genes (PCNA,CDK1,CCND2),antioxidant genes (DHODH,GPX4,AIFM2),and anti-apoptotic genes (BCL-2),while significantly promoting the expression of apoptotic genes (CAS-3,BAX).Notably,0.10 μmol/L ZEN obviously promoted the expression of milk fat synthesis-related genes (PPARγ,FASN,JAK-2),but 10.00 μmol/L ZEN significantly suppressed these genes.The aforementioned results suggested different effects of ZEN concentrations on MAC-T cells:0.10 μmol/L ZEN could promote MAC-T cell growth and milk fat synthesis-related gene expression while also inducing the expression of apoptotic genes.In contrast,10.00 μmol/L ZEN induces oxidative stress in MAC-T cells,reduces mitochondria number,inhibits the expression of genes associated with proliferation,antioxidant properties,anti-apoptotic properties,and milk fat synthesis,while promoting the expression of apoptotic genes,leading to cell apoptosis.

Key words: Milk, Zearalenone, Bovine mammary epithelial cells, Milk fat synthesis, Proliferation, Apoptosis

马梓峰, 李巧, 徐红梅, 李悦悦, 殷实, 何翃闳, 熊燕, 兰道亮, 李键, 熊显荣, 付伟. 玉米赤霉烯酮对奶牛乳腺上皮细胞生长和乳脂合成相关基因表达的影响[J]. 华北农学报, 2024, 39(1): 201-210. doi: 10.7668/hbnxb.20194501.

MA Zifeng, LI Qiao, XU Hongmei, LI Yueyue, YIN Shi, HE Honghong, XIONG Yan, LAN Daoliang, LI Jian, XIONG Xianrong, FU Wei. The Effect of Zearalenone on the Growth of Bovine Mammary Epithelial Cells and the Expression of Genes Related to Milk Fat Synthesis[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(1): 201-210. doi: 10.7668/hbnxb.20194501.

http://www.hbnxb.net/CN/Y2024/V39/I1/201

图/表 11

表1 RT-qPCR引物序列

Tab.1 Primers for RT-qPCR

基因 Gene	引物序列(5'—3') Primer sequences(5'—3')	NCBI 序列号 NCBI sequence ID	产物长度/bp Product size	退火温度/℃ Tm
GAPDH	F:ACAGTCAAGGCAGAGAACGG R:GGTTCACGCCCATCACAAAC	XM_027541111.1	235	60
GPX4	F:TGTGGTTTACGGATCCTGGC R:CCCTTGGGCTGGACTTTCAT	NM_001346431.1	182	60
JAK-2	F:GAGGCACCGGTTTCAGAAG R:AGCCATTCCCATGCAGAGTC	XM_024986698.1	208	60
DHODH	F:GGAGCTGCGTCCTTGATTCT R:TTCTAGGCAAGAGGGTTGCG	NM_001015650.1	141	60
PPARγ	F:GATAGGTGTGATCTTAACTGTCGGAT R:CGCTAACAGCTTCTCCTTCTCG	NM_181024.2	150	60
FASN	F:CTGGAGCGTGAGCACAACCTG R:GTGTGGAGTCCGTCAGCTCAT	XM_005220997.3	118	60
BAX	F:GTGCCCGAGTTGATCAGGAC R:CCATGTGGGTGTCCCAAAGT	XM_015458140.2	175	60
BCL-2	F:GAGTTCGGAGGGGTCATGTG R:GCCTTCAGAGACAGCCAGGA	NM_001166486.1	203	60
PCNA	F:CCTTGGTGCAGCTAACCCTT R:TTGGACATGCTGGTGAGGTT	NM_001034494.1	94	60
CCND2	F:CACCGATGTGGATTGCCTCA R:TCCAGCTCATCCTCCGACTT	NM_001076372.1	117	60
CDK1	F:GCACACAATATGGACCCCCA R:CTGAAGCCTCAGAGCCACTC	NM_001012669.1	139	60
AIFM2	F:TGGCCATGGGGAGAAATGAC R:TTTTCCAGCTCGTGGAGACC	NM_001314031.1	111	60
CAS-3	F:CGAGGCACAGAACTGGACTG R:GCCAGGAAAAGTAACCAGGTG	NM_001077840.1	130	60

图1 CCK-8检测ZEN对MAC-T细胞活力的影响不同小写字母表示差异显著(P<0.05),不同大写字母表示差异极显著(P<0.01)。图3,5,7—10同。

Fig.1 The influence of ZEN on the viability of MAC-T cells assessed through CCK-8 assay Different lowercase letters indicate significant differences(P<0.05), different capital letters indicate extremely significant differences(P<0.01).The same as Fig.3,5,7—10.

图 2 光学显微镜下观察ZEN对MAC-T细胞生长状态的影响

Fig.2 Light microscope observe the effects of ZEN on the growth of MAC-T cells

图3 不同浓度ZEN对MAC-T细胞数量的影响

Fig.3 Effect of ZEN on the number of MAC-T cell

图4 ZEN处理后MAC-T细胞凋亡和坏死情况分析红色方框表示图片部分放大区域,Magnification表示局部放大图像。图6同。

Fig.4 Apoptosis and necrosis analysis of MAC-T cells after ZEN treatment Red box means enlarged view of local area in image,Magnification means locally enlarged image.The same as Fig.6.

图5 ZEN对MAC-T细胞内ROS水平影响

Fig.5 Effect of ZEN on ROS level in MAC-T cells

图6 ZEN对MAC-T细胞线粒体数量和分布的影响

Fig.6 Effect of ZEN on mitochondria number and distribution in MAC-T cells

图7 增殖相关基因表达量分析

Fig.7 Expression analysis of genes related to proliferation

图8 凋亡相关基因表达量分析

Fig.8 Expression analysis of genes related to apoptosis

图9 氧化应激相关基因表达量分析

Fig.9 Expression analysis of genes related to oxidative stress

图10 乳脂合成相关基因表达量分析

Fig.10 Expression analysis of genes involved in milk fat synthesis

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玉米赤霉烯酮对奶牛乳腺上皮细胞生长和乳脂合成相关基因表达的影响

The Effect of Zearalenone on the Growth of Bovine Mammary Epithelial Cells and the Expression of Genes Related to Milk Fat Synthesis

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