DNA甲基化对LPS诱导奶牛子宫内膜细胞IL-6、IL-8表达的作用研究

发布时间：2018-04-11 03:35

本文选题：LPS + 奶牛子宫内膜细胞　；参考：《吉林农业大学》2017年硕士论文

【摘要】：奶牛子宫内膜炎是奶牛产后细菌感染子宫而引起的产科疾病,严重影响奶牛业的健康发展。以往对其研究多集中于基因的表达量变化,但其作用机制报道较少。本研究着力于阐明细菌脂多糖(lipopolysaccharide,LPS)影响白细胞介素6(interleukin-6,IL-6)和白细胞介素8(interleukin-8,IL-8)表达的作用机制,首先利用1μg/m LLPS刺激奶牛子宫内膜细胞24h,以此为基础研究DNA甲基化转移相关酶(Me CP2、DNMT1、DNMT3A和DNMT3B)的表达变化及DNA甲基化转移酶抑制剂(5-aza-2'-deoxycytidine,5Aza-dc)对IL-6和IL-8表达的影响,最后利用亚硫酸氢测序(bisulfite sequencing PCR,BSP)技术分析LPS对IL-6和IL-8启动子区甲基化状态的影响。研究结果如下:1、利用牛子宫内膜组织块培养法获得奶牛子宫内膜基质细胞和上皮细胞共培养体系,用1μg/m LLPS处理细胞24h,ELISA法检测发现LPS可显著促进细胞IL-6和IL-8的蛋白分泌量(p0.01),荧光定量PCR检测发现LPS可显著提高细胞IL-6和IL-8的m RNA表达量(p0.01)。2、首先研究LPS处理细胞24h后,MeCP2、DNMT1、DNMT3A和DNMT3B的mRNA表达变化,结果表明,LPS可显著增加Me CP2、DNMT1、DNMT3A和DNMT3B的m RNA表达量(p0.01)。随后研究不同浓度(1μmol/L、2μmol/L、5μmol/L和10μmol/L)5Aza-dc分别处理不同时间(24h、48h和72h)后,LPS诱导的奶牛子宫内膜细胞IL-6和IL-8的m RNA表达变化,结果表明,5Aza-dc可显著提高IL-6和IL-8的m RNA表达量(p0.01)。3、利用BSP技术研究LPS对IL-6和IL-8启动子区甲基化状态的影响。结果表明,LPS可降低IL-6和IL-8启动子区甲基化率,与对照组相比,IL-6启动子区甲基化率下降2%,IL-8启动子区甲基化率下降10.4%。其中,IL-6启动子区-660及IL-8启动子区-120和-48位点甲基化程度明显低于对照组。本研究利用1μg/mL LPS刺激奶牛子宫内膜细胞24h分析IL-6和IL-8表达机制,研究结果表明,DNA甲基化通过降低IL-6和IL-8的启动子区甲基化水平来参与LPS诱导的奶牛子宫内膜细胞IL-6和IL-8的表达过程。
[Abstract]:Endometritis is an obstetrical disease caused by postpartum bacterial infection in dairy cows, which seriously affects the healthy development of dairy cattle industry.In the past, most of the studies focused on the changes of gene expression, but the mechanism of its action was less reported.The purpose of this study was to elucidate the mechanism of the effect of lipopolysaccharide (LPSs) on the expression of interleukin-6 (IL-6) and interleukin-8 (IL-8).At first, 1 渭 g / m LLPS was used to stimulate endometrial cells for 24 h, based on which we studied the expression of DNA methylation transfer related enzymes (me CP2DNMT1, DNMT3A and DNMT3B) and the effect of DNA methyltransferase inhibitor (DNA 5-aza-2) -deoxycytidine 5Aza-dc) on the expression of IL-6 and IL-8.Finally, the effect of LPS on the methylation of IL-6 and IL-8 promoter region was analyzed by bisulfite sequencing BSPs technique.The results are as follows: 1. The co-culture system of bovine endometrial stromal cells and epithelial cells was obtained by using the method of bovine endometrial tissue mass culture.And the change of mRNA expression in DNMT3B,The results showed that lipopolysaccharide could significantly increase the m RNA expression of DNMT3A and DNMT3B.Then the changes of m RNA expression of IL-6 and IL-8 in bovine endometrial cells induced by LPS-induced treatment with different concentrations of 1 渭 mol / L ~ 2 渭 mol / L ~ (2 渭 mol / L) 5 渭 mol/L and 10 渭 mol/L)5Aza-dc for 24 h and 72 h, respectively, were studied.The results showed that 5 Aza-dc could significantly increase the expression of m RNA in IL-6 and IL-8. The effect of LPS on the methylation of IL-6 and IL-8 promoter was studied by BSP technique.The results showed that lipopolysaccharide could decrease the methylation rate of IL-6 and IL-8 promoter. Compared with the control group, the methylation rate of IL-6 promoter was decreased by 2% and the methylation rate of IL-8 promoter was decreased by 10.4%.The methylation degree of -660 and -120 and -48 in the IL-6 promoter and IL-8 promoter was significantly lower than that in the control group.In this study, 1 渭 g/mL LPS was used to stimulate the expression of IL-6 and IL-8 in dairy cow endometrial cells for 24 hours.The results showed that DNA methylation was involved in the expression of IL-6 and IL-8 in bovine endometrial cells induced by LPS by reducing the promoter methylation level of IL-6 and IL-8.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S858.23

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