PCYOX1L基因调控LPS诱导的牛子宫内膜上皮细胞的功能研究  

Study on the Function of PCYOX 1 L Gene Regulating LPS-induced Bovine Endometrial Epithelial Cells

作　　者：冀国尚 盛辉 张俊星 冯雪 户春丽 王雅春 马燕芬 李莉 杨文飞 马云 JI Guoshang;SHENG Hui;ZHANG Junxing;FENG Xue;HU Chunli;WANG Yachun;MA Yanfen;LI Li;YANG Wenfei;MA Yun(Key Laboratory of Molecular Cell Breeding of Ningxia Hui Autonomous Region,College of Animal Science and Technology,Ningxia University,Yinchuan 750000,China;Ningxia Animal Disease Prevention and Control Center,Yinchuan 750000,China;Ningxia Xin’ao Agriculture and Animal Husbandry Co.,Ltd.,Lingwu 751400,China)

机构地区：[1]宁夏大学动物科技学院,宁夏回族自治区分子细胞育种重点实验室,银川750000 [2]宁夏动物疾病预防控制中心,银川750000 [3]宁夏新澳农牧有限公司,灵武751400

出　　处：《中国畜牧兽医》2024年第7期2984-2997,共14页China Animal Husbandry & Veterinary Medicine

基　　金：现代农业产业技术体系(CARS-36);宁夏回族自治区重点研发计划项目(2022BBF02017、2021BEF01001);中央引导地方科技发展专项(YDZX2022153)。

摘　　要：【目的】探究异戊二烯基半胱氨酸氧化酶-1样蛋白(prenylcysteine oxidase 1 like protein,PCYOX1L)对脂多糖(lipopolysaccharide,LPS)诱导的牛子宫内膜上皮细胞(bovine endometrial epithelial cells,BEECs)的炎症、增殖和凋亡的调控作用,以期明确PCYOX1L基因对奶牛子宫内膜炎发生的调控机制。【方法】利用LPS刺激BEECs构建体外炎症模型,设计合成PCYOX1L基因的小干扰RNA(si-PCYOX1L)和过表达质粒载体(pcDNA3.1-PCYOX1L),采用Lipofectamine 3000转染试剂将si-PCYOX1L和pcDNA3.1-PCYOX1L转染至BEECs,通过实时荧光定量PCR检测干扰和过表达PCYOX1L基因对细胞炎症、增殖和凋亡标志基因mRNA表达水平的影响,利用ELISA方法检测白细胞介素-1(IL-1)的蛋白表达水平。利用试剂盒检测BEECs中活性氧(ROS)含量,并采用EdU、CCK-8和流式细胞术检测细胞活力、增殖及周期。利用线粒体膜电位试剂盒检测细胞线粒体损伤,并通过流式细胞术检测细胞凋亡情况。【结果】试验成功构建pcDNA3.1-PCYOX1L过表达载体,并筛选出si-PCYOX1L-385的干扰效果最佳。与对照组相比,干扰PCYOX1L基因后,炎症标志基因(IL-1β、IL-6、IL-8)mRNA表达量极显著下调(P<0.01),IL-1蛋白含量显著降低(P<0.05),细胞内ROS水平极显著降低(P<0.01);增殖标志基因(CDK2、CDK4、PCNA、CCND2)mRNA表达量极显著或显著上调(P<0.01;P<0.05),细胞活力上升,促进细胞从S期向G2期的转变;促凋亡基因Bax表达水平显著降低(P<0.05),抑凋亡基因BCL2表达水平上升,极显著降低了BEECs凋亡率(P<0.01)。过表达PCYOX1L基因后结果与干扰PCYOX1L基因后结果相反。【结论】PCYOX1L基因促进了BEECs炎症的发生,抑制细胞增殖,并促进细胞凋亡。本研究结果为进一步探究PCYOX1L基因调控奶牛子宫内膜炎发生的分子机制提供基础数据。【Objective】This study was aimed to investigate the regulatory effects of prenylcysteine oxidase 1 like protein(PCYOX1L)on lipopolysaccharide(LPS)-induced inflammation,proliferation,and apoptosis in bovine endometrial epithelial cells(BEECs),so as to clarify the regulatory mechanism of PCYOX 1 L gene on the occurrence of endometritis in dairy cows.【Method】An in vitro inflammation model was constructed by stimulating BEECs with LPS,and small interfering RNA(si-PCYOX1L)and overexpression vector(pcDNA3.1-PCYOX1L)of PCYOX 1 L gene were designed and synthesized,Lipofectamine 3000 transfection reagent was used to transfect the BEECs with pcDNA3.1-PCYOX1L.The effects of interference and overexpression of PCYOX 1 L gene on the mRNA expression of cellular inflammation,proliferation and apoptosis marker genes were detected by Real-time quantitative PCR.Reactive oxygen species(ROS)level in cell was detected by kit,the protein expression of interleukin-1(IL-1)was detected by ELISA method,and the cell viability,proliferation and cycle were detected by EdU,CCK-8 and flow cytometry.Cellular mitochondrial damage was detected by a mitochondrial membrane potential kit,and the apoptosis was detected by flow cytometry.【Result】pcDNA3.1-PCYOX1L overexpression vector was successfully constructed in this experiment,and the interference effect of si-PCYOX1L-385 was screened out to be the best.Compared with control group,after interfering with PCYOX 1 L gene,the mRNA expression of inflammatory marker genes(IL-1β,IL-6 and IL-8)was extremely significantly down-regulated(P<0.01),the IL-1 protein content was significantly increased(P<0.05),and the intracellular ROS level was extremely significantly decreased(P<0.01).The mRNA expressions of proliferative marker genes(CDK2,CDK4,PCNA and CCND2)were extremely significantly or significantly up-regulated(P<0.01 or P<0.05),and the cell viability was increased,promoting the transition of cells from S phase to G2 phase.The expression of pro-apoptotic gene Bax was significantly decreased(P<

关 键 词：奶牛 子宫内膜炎 PCYOX1L基因 细胞增殖 细胞凋亡 

分 类 号：S814.8[农业科学—畜牧学]