植物雌激素大豆黄酮对牛乳腺上皮细胞增殖及细胞周期的影响  

Effects of Phytoestrogen Daidzein on Proliferation and Cell Cycle of Bovine Mammary Epithelial Cells

作　　者：谢娜娜 闫书平 张崇昊 曹西月 张源淑[1] XIE Nana;YAN Shuping;ZHANG Chonghao;CAO Xiyue;ZHANG Yuanshu(Key Laboratory of Animal Physiology and Biochemistry,Ministry of Agriculture,Nanjing Agriculture University,Nanjing 210095,China)

机构地区：[1]南京农业大学动物医学院,农业部动物生理生化重点开放实验室,南京210095

出　　处：《动物营养学报》2022年第4期2645-2653,共9页CHINESE JOURNAL OF ANIMAL NUTRITION

基　　金：国家自然科学基金(31972640)。

摘　　要：本试验采用大豆黄酮(DZ)作用于牛乳腺上皮MAC-T细胞,探讨其通过上调雌激素受体β(ERβ)表达对细胞体外增殖及细胞周期的影响,为DZ改善乳腺发育提供依据。采用不同浓度DZ(0~160.0μmol/L)处理MAC-T细胞24 h,通过检测细胞活力确定DZ作用浓度;然后采用不同浓度DZ(0、2.5、5.0和10.0μmol/L)处理MAC-T细胞12、24、36和48 h,通过检测细胞活力确定DZ作用时间;再用生理剂量的雌二醇(E2)作为阳性对照,对各处理细胞进行细胞计数,采用Western blot分析细胞内细胞周期相关蛋白、ERβ蛋白相对表达量,并用流式细胞技术分析细胞周期变化。结果表明:1)与对照组相比,2.5和5.0μmol/L DZ处理细胞均能提高MAC-T细胞活力,其中5.0μmol/L DZ处理显著提高细胞活力(P<0.05);而在浓度高于10.0μmol/L后,细胞活力均有下降,因此确定了DZ作用的低、中和高浓度分别为2.5、5.0和10.0μmol/L;而与对照组相比,处理12 h后,5.0μmol/L DZ处理使得细胞活力显著提高(P<0.05),其他2个浓度处理对细胞活力无显著影响(P>0.05),因此确认DZ作用时间为12 h。2)与对照组相比,5.0μmol/L DZ处理使得细胞数量显著增加(P<0.05);2.5μmol/L DZ处理使得细胞数量有所增加,但差异不显著(P>0.05);E2处理使得细胞数量略低于各DZ处理,但差异不显著(P>0.05)。3)Western blot结果显示,与对照组相比,3个浓度DZ处理均可上调增殖细胞核抗原(PCNA)、细胞周期蛋白D1(CyclinD1)、细胞周期蛋白D3(CyclinD3)和ERβ的蛋白相对表达量,其中5.0μmol/L DZ处理显著上调PCNA、CyclinD1和ERβ的蛋白相对表达量(P<0.05)。4)与对照组相比,5.0μmol/L DZ处理极显著提高了G2-M期细胞占比(P<0.01),同时极显著提高了S期细胞占比(P<0.01);E2处理与5.0μmol/L DZ处理趋势相同,均极显著提高了G2-M期细胞占比(P<0.01),同时提高了S期细胞占比(P>0.05)。结果提示,植物雌激素DZ具有类雌激素样作用,可通过上调ERβ蛋白表达,提高G2-M�In this study,daidzein(DZ)was applied to bovine mammary epithelial MAC-T cells to investigate the effects of DZ on cell proliferation and cell cycle by up-regulating the expression of estrogen receptorβ(ERβ),providing evidence for DZ to improve mammary gland development.MAC-T cells were treated with different concentrations of DZ(0 to 160.0μmol/L)for 24 h,and the concentration of DZ was determined by detecting cell viability.Then MAC-T cells were treated with different concentrations of DZ(0,2.5,5.0 and10.0μmol/L)for 12,24,36 and 48 h,and the time of DZ action was determined by detecting cell viability.The physiological dose of estradiol(E2)was used as positive control,and the cells were counted.Western blot was used to analyze the relative expression levels of cyclin-related proteins and ERβprotein,and flow cytometry was used to analyze the changes of cell cycle.The results showed as follows:1)compared with the control group,both 2.5 and 5.0μmol/L DZ treatments could improve the cell viability of MAC-T cells,and5.0μmol/L DZ treatment significantly increased the cell viability(P<0.05).The cell viability decreased when the concentration was higher than 10.0μmol/L.Therefore,the low,medium and high concentrations of DZ were determined to be 2.5,5.0 and 10.0μmol/L,respectively.Compared with the control group,5.0μmol/L DZ treatment significantly increased the cell viability after 12 h treatment(P<0.05),while the other two concentrations had no significant effect on cell viability(P>0.05).Therefore,the effect time of DZ was confirmed to be 12 h.2)Compared with the control group,5.0μmol/L DZ treatment significantly increased cell number(P<0.05);the cell number was increased by 2.5μmol/L DZ treatment,but the difference was not significant(P>0.05);the cell number of E2 treatment was slightly lower than that of DZ treatments,but the difference was not significant(P>0.05).3)Western blot results showed that compared with the control group,the relative protein expression levels of proliferating nuclear antigen(PCN

关 键 词：MAC-T细胞 植物雌激素 大豆黄酮 细胞增殖 细胞周期 

分 类 号：S816[农业科学—饲料科学]