本文選題：LPS + 奶牛子宮內(nèi)膜細胞　； 參考：《吉林農(nóng)業(yè)大學》2017年碩士論文

【摘要】：奶牛子宮內(nèi)膜炎是奶牛產(chǎn)后細菌感染子宮而引起的產(chǎn)科疾病,嚴重影響奶牛業(yè)的健康發(fā)展。以往對其研究多集中于基因的表達量變化,但其作用機制報道較少。本研究著力于闡明細菌脂多糖(lipopolysaccharide,LPS)影響白細胞介素6(interleukin-6,IL-6)和白細胞介素8(interleukin-8,IL-8)表達的作用機制,首先利用1μg/m LLPS刺激奶牛子宮內(nèi)膜細胞24h,以此為基礎(chǔ)研究DNA甲基化轉(zhuǎn)移相關(guān)酶(Me CP2、DNMT1、DNMT3A和DNMT3B)的表達變化及DNA甲基化轉(zhuǎn)移酶抑制劑(5-aza-2'-deoxycytidine,5Aza-dc)對IL-6和IL-8表達的影響,最后利用亞硫酸氫測序(bisulfite sequencing PCR,BSP)技術(shù)分析LPS對IL-6和IL-8啟動子區(qū)甲基化狀態(tài)的影響。研究結(jié)果如下:1、利用牛子宮內(nèi)膜組織塊培養(yǎng)法獲得奶牛子宮內(nèi)膜基質(zhì)細胞和上皮細胞共培養(yǎng)體系,用1μg/m LLPS處理細胞24h,ELISA法檢測發(fā)現(xiàn)LPS可顯著促進細胞IL-6和IL-8的蛋白分泌量(p0.01),熒光定量PCR檢測發(fā)現(xiàn)LPS可顯著提高細胞IL-6和IL-8的m RNA表達量(p0.01)。2、首先研究LPS處理細胞24h后,MeCP2、DNMT1、DNMT3A和DNMT3B的mRNA表達變化,結(jié)果表明,LPS可顯著增加Me CP2、DNMT1、DNMT3A和DNMT3B的m RNA表達量(p0.01)。隨后研究不同濃度(1μmol/L、2μmol/L、5μmol/L和10μmol/L)5Aza-dc分別處理不同時間(24h、48h和72h)后,LPS誘導(dǎo)的奶牛子宮內(nèi)膜細胞IL-6和IL-8的m RNA表達變化,結(jié)果表明,5Aza-dc可顯著提高IL-6和IL-8的m RNA表達量(p0.01)。3、利用BSP技術(shù)研究LPS對IL-6和IL-8啟動子區(qū)甲基化狀態(tài)的影響。結(jié)果表明,LPS可降低IL-6和IL-8啟動子區(qū)甲基化率,與對照組相比,IL-6啟動子區(qū)甲基化率下降2%,IL-8啟動子區(qū)甲基化率下降10.4%。其中,IL-6啟動子區(qū)-660及IL-8啟動子區(qū)-120和-48位點甲基化程度明顯低于對照組。本研究利用1μg/mL LPS刺激奶牛子宮內(nèi)膜細胞24h分析IL-6和IL-8表達機制,研究結(jié)果表明,DNA甲基化通過降低IL-6和IL-8的啟動子區(qū)甲基化水平來參與LPS誘導(dǎo)的奶牛子宮內(nèi)膜細胞IL-6和IL-8的表達過程。
[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.
【學位授予單位】：吉林農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S858.23

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