PCYOX1L基因调控LPS诱导的牛子宫内膜上皮细胞的功能研究

doi:10.16431/j.cnki.1671-7236.2024.07.024

摘要/Abstract

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。

关键词: 奶牛; 子宫内膜炎; PCYOX1L基因; 细胞增殖; 细胞凋亡

Abstract: 【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 PCYOX1L 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 PCYOX1L 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 PCYOX1L 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 PCYOX1L 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＜0.05),and the expression of anti-apoptotic gene BCL2 was increased,reducing the apoptosis rate of BEECs(P＜0.01).However,the results were exactly the opposite after overexpression of PCYOX1L gene.【Conclusion】 PCYOX1L gene promoted inflammation,inhibited cell proliferation,and promoted apoptosis in BEECs.This results provided basic data for further investigation of the molecular mechanism of PCYOX1L gene regulating the occurrence of endometritis in dairy cows.

Key words: dairy cows; endometritis; PCYOX1L gene; cell proliferation; apoptosis

中图分类号:

S814.8

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. PCYOX1L基因调控LPS诱导的牛子宫内膜上皮细胞的功能研究[J]. 中国畜牧兽医, 2024, 51(7): 2984-2997.

JI Guoshang, SHENG Hui, ZHANG Junxing, FENG Xue, HU Chunli, WANG Yachun, MA Yanfen, LI Li, YANG Wenfei, MA Yun. Study on the Function of PCYOX1L Gene Regulating LPS-induced Bovine Endometrial Epithelial Cells[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(7): 2984-2997.

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