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内毒素诱导血管内皮细胞NF-κB-Jmjd3信号轴下游基因表达的表观遗传学调控机制

发布时间:2019-06-03 23:53
【摘要】:目的:慢性炎症与肿瘤关系密切,血管内皮细胞在炎症和肿瘤的病理过程中发挥重要作用。本研究以内毒素(LPS)激活血管内皮细胞,释放炎症介质和粘附分子,探索内皮细胞NF-κB-Jmjd3信号轴在内皮细胞炎症反应过程的的作用,并分析其可能的表观遗传学调控机制,为有效抑制血管炎症反应,寻找相关疾病治疗靶点,提供实验依据。方法:1.细胞培养及分组:复苏并常规培养原代人脐静脉内皮细胞(HUVECs),随机分为对照组与LPS实验组,实验组按照不同检测指标要求,予LPS刺激不同时间段后取样待测。2、LPS诱导内皮细胞炎性介质和粘附分子的表达释放:ELISA试剂盒检测细胞上清液中IL-6、MMP-9和ICAM-1与表达变化;3、NF-κB/p65、Jmjd3的表达与定位:免疫荧光实验观察NF-κB/p65、Jmjd3的表达与定位变化;RT-PCR检测Jmjd3 RNA水平的表达变化;蛋白质免疫印迹法验证NF-κB/p65,IκBα和Jmjd3的表达变化;4、NF-κB/p65转录因子活性测定:Millipore转录因子试剂盒检测胞核NF-κB/p65蛋白活性;5、染色质免疫共沉淀技术分析目标基因的转录起始结合位点区域NF-κB/p65、Jmjd3、H3K27me3的募集关系:应用Millipore染色质免疫共沉淀试剂盒建立目标DNA文库,Ch IP-q PCR分析在目标基因IL-6、MMP-9和ICAM-1转录起始结合位点NF-κB/p65、Jmjd3、H3K27me3的募集关系。结果:1、LPS诱导内皮细胞炎性介质和粘附分子的表达释放:细胞上清液中IL-6、MMP-9和ICAM-1分别与相应的对照组相比表达显著升高,差异均具有统计学意义(P0.01)。2、NF-κB-Jmjd3信号通路的活化:(1)NF-κB/p65免疫荧光实验中LPS各组与空白对照组(0h组)相比,随着刺激时间增加整体荧光强度逐渐增加,NF-κB/p65表达上调,并逐渐向转胞核内聚集,核转位高峰为2 h。(2)NF-κB/p65转录因子活性测定:LPS刺激2h组中NF-κB/p65蛋白相对活性显著高于对照组(0h组)(P0.01),余各实验组与对照组相比均无显著差异(P0.05)。(3)RT-PCR检测Jmjd3 RNA水平变化:LPS 2h组RNA水平显著高于对照组(0h组),差异有统计学意义(P0.01),余各实验组与对照组相比均无显著差异(P0.05)。(4)Jmjd3免疫荧光实验中LPS各组与对照组(0h组)相比Jmjd3的表达均上调,其中于1h和2h上调明显。(5)蛋白质免疫印迹法验证总NF-κB/p65蛋白随着LPS刺激时间增加表达逐渐上调;胞核NF-κB/p65蛋白于2h和6h表达明显上调;IκBα蛋白于LPS 2h和4h组表达明显下调;Jmjd3表达亦上调,于1h和2h上调明显。(6)染色质免疫共沉淀技术分析目标基因转录起始结合位点NF-κB/p65、Jmjd3和H3K27me3的募集关系:NF-κB/p65关联DNA文库中IL-6、MMP-9的相对扩增量与相应的对照组相比显著升高(P0.01),而ICAM-1与相应对照组相比升高,但差异无统计学意义(P0.05);Jmjd3关联DNA文库、H3K27me3关联DNA文库中目标基因扩增结果与NF-κB/p65关联DNA文库扩增结果一致。结论:LPS诱导内皮细胞中NF-κB-Jmjd3信号通路激活,活化高峰时间为2h:NF-κB信号通路活化后释放转录因子入核结合到Jmjd3相应启动子区域,调控Jmjd3表达,上调的Jmjd3入核作用于目标基因区域组蛋白H3K27me3使其去甲基化、改变构象,从而暴露目标基因转录起始序列,NF-κB通路的活化时也调控转录因子本身表达,上调的核因子-κB结合到暴露的转录起始序列上调控目标基因的转录,引起炎症介质和粘附分子的表达影响内皮细胞微环境,导致炎症相关病理过程。
[Abstract]:Objective: Chronic inflammation is closely related to the tumor, and the vascular endothelial cells play an important role in the pathological process of inflammation and tumor. In this study, endothelial cells were activated by endotoxin (LPS), the inflammatory mediators and adhesion molecules were released, and the role of the NF-B-Jmjd3 signal axis in the inflammatory reaction of the endothelial cells was explored, and the possible epigenetic control mechanism was analyzed to effectively inhibit the inflammatory reaction of the blood vessel. Find relevant disease treatment target and provide experimental basis. Method:1. Cell culture and grouping: resuscitation and routine culture of primary human umbilical vein endothelial cells (HUVECs) were randomly divided into control group and LPS group. The expression and release of IL-6, MMP-9 and ICAM-1 in the supernatant of the cells were detected by ELISA, and the expression and localization of NF-B/ p65 and Jmjd3 were observed by immunofluorescence. The expression of Jmjd3 RNA was detected by RT-PCR, and the expression of NF-B/ p65, I, B and Jmjd3 was verified by Western blot. The relationship between the transcription initiation binding site region NF-B/ p65, Jmjd3, and H3K27me3 of the target gene is analyzed by the chromatin immunoprecipitation technique: the target DNA library is established by using the Millipore chromatin immunoprecipitation kit, and the Ch IP-q PCR is analyzed on the target gene IL-6, The relationship between the initial binding sites of MMP-9 and ICAM-1 and the recruitment of NF-B/ p65, Jmjd3, and H3K27me3. Results:1. The expression of IL-6, MMP-9 and ICAM-1 in the supernatant of the cells was significantly higher than that of the corresponding control group (P0.01). (1) Compared with the blank control group (0h group), the expression of NF-EMAB/ p65 was up-regulated with the increase of the stimulation time, and the expression of NF-EMAB/ p65 was up-regulated and gradually increased to the nucleus of the subcellular nucleus, compared with the control group (0 h group). (2) The relative activity of NF-B/ p65 protein was significantly higher than that in the control group (group 0 h) (P 0.01), and there was no significant difference between the experimental group and the control group (P0.05). (3) The level of the mRNA of Jmjd3 was detected by RT-PCR: the level of RNA in the LPS group was significantly higher than that of the control group (group 0h), the difference was significant (P0.01), and there was no significant difference between the experimental group and the control group (P0.05). (4) The expression of Jmjd3 was up-regulated in the group of LPS and control group (group 0h), and the expression of Jmjd3 was up-regulated at 1 h and 2 h. (5) The expression of NF-EMAB/ p65 was up-regulated with the increase of LPS-stimulated time, and the expression of NF-EMAB/ p65 was up-regulated in 2h and 6h, and the expression of Jmjd3 was up-regulated, and the expression of Jmjd3 was up-regulated at 1 h and 2 h. (6) The relationship between the expression of NF-B/ p65, Jmjd3 and H3K27me3 in the transcription initiation binding site of the target gene was analyzed by the chromatin immunoprecipitation technique: the relative increase of IL-6 and MMP-9 in the DNA library associated with NF-B/ p65 was significantly higher than that of the corresponding control group (P0.01), while the ICAM-1 was increased compared with the corresponding control group. The results of the amplification of the target gene in the associated DNA library of the Jmjd3-associated DNA library and the H3K27me3-associated DNA library were consistent with the results of the amplification of the DNA library associated with the NF-B/ p65. Conclusion: The activation of NF-B-Jmjd3 signal pathway in the endothelial cells was induced by LPS, and the activation peak time was 2 h. After the activation of NF-B-B signal pathway, the transcription factor was released into the region of the corresponding promoter of Jmjd3, and the expression of Jmjd3 was regulated. The up-regulated Jmjd3 entry nucleus acts on the target gene region histone H3K27me3 to demethylation and change the conformation, so that the transcription initiation sequence of the target gene and the activation of the NF-HACB pathway also regulate the expression of the transcription factor itself, The up-regulated nuclear factor-B is bound to the exposed transcription initiation sequence to regulate the transcription of the target gene, and the expression of the inflammatory mediator and the adhesion molecule affects the microenvironment of the endothelial cells and leads to an inflammation-related pathological process.
【学位授予单位】:南昌大学
【学位级别】:硕士
【学位授予年份】:2015
【分类号】:R730.2

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