內(nèi)毒素誘導血管內(nèi)皮細胞NF-κB-Jmjd3信號軸下游基因表達的表觀遺傳學調(diào)控機制
發(fā)布時間：2019-06-03 23:53
【摘要】：目的:慢性炎癥與腫瘤關(guān)系密切,血管內(nèi)皮細胞在炎癥和腫瘤的病理過程中發(fā)揮重要作用。本研究以內(nèi)毒素(LPS)激活血管內(nèi)皮細胞,釋放炎癥介質(zhì)和粘附分子,探索內(nèi)皮細胞NF-κB-Jmjd3信號軸在內(nèi)皮細胞炎癥反應(yīng)過程的的作用,并分析其可能的表觀遺傳學調(diào)控機制,為有效抑制血管炎癥反應(yīng),尋找相關(guān)疾病治療靶點,提供實驗依據(jù)。方法:1.細胞培養(yǎng)及分組:復蘇并常規(guī)培養(yǎng)原代人臍靜脈內(nèi)皮細胞(HUVECs),隨機分為對照組與LPS實驗組,實驗組按照不同檢測指標要求,予LPS刺激不同時間段后取樣待測。2、LPS誘導內(nèi)皮細胞炎性介質(zhì)和粘附分子的表達釋放:ELISA試劑盒檢測細胞上清液中IL-6、MMP-9和ICAM-1與表達變化;3、NF-κB/p65、Jmjd3的表達與定位:免疫熒光實驗觀察NF-κB/p65、Jmjd3的表達與定位變化;RT-PCR檢測Jmjd3 RNA水平的表達變化;蛋白質(zhì)免疫印跡法驗證NF-κB/p65,IκBα和Jmjd3的表達變化;4、NF-κB/p65轉(zhuǎn)錄因子活性測定:Millipore轉(zhuǎn)錄因子試劑盒檢測胞核NF-κB/p65蛋白活性;5、染色質(zhì)免疫共沉淀技術(shù)分析目標基因的轉(zhuǎn)錄起始結(jié)合位點區(qū)域NF-κB/p65、Jmjd3、H3K27me3的募集關(guān)系:應(yīng)用Millipore染色質(zhì)免疫共沉淀試劑盒建立目標DNA文庫,Ch IP-q PCR分析在目標基因IL-6、MMP-9和ICAM-1轉(zhuǎn)錄起始結(jié)合位點NF-κB/p65、Jmjd3、H3K27me3的募集關(guān)系。結(jié)果:1、LPS誘導內(nèi)皮細胞炎性介質(zhì)和粘附分子的表達釋放:細胞上清液中IL-6、MMP-9和ICAM-1分別與相應(yīng)的對照組相比表達顯著升高,差異均具有統(tǒng)計學意義(P0.01)。2、NF-κB-Jmjd3信號通路的活化:(1)NF-κB/p65免疫熒光實驗中LPS各組與空白對照組(0h組)相比,隨著刺激時間增加整體熒光強度逐漸增加,NF-κB/p65表達上調(diào),并逐漸向轉(zhuǎn)胞核內(nèi)聚集,核轉(zhuǎn)位高峰為2 h。(2)NF-κB/p65轉(zhuǎn)錄因子活性測定:LPS刺激2h組中NF-κB/p65蛋白相對活性顯著高于對照組(0h組)(P0.01),余各實驗組與對照組相比均無顯著差異(P0.05)。(3)RT-PCR檢測Jmjd3 RNA水平變化:LPS 2h組RNA水平顯著高于對照組(0h組),差異有統(tǒng)計學意義(P0.01),余各實驗組與對照組相比均無顯著差異(P0.05)。(4)Jmjd3免疫熒光實驗中LPS各組與對照組(0h組)相比Jmjd3的表達均上調(diào),其中于1h和2h上調(diào)明顯。(5)蛋白質(zhì)免疫印跡法驗證總NF-κB/p65蛋白隨著LPS刺激時間增加表達逐漸上調(diào);胞核NF-κB/p65蛋白于2h和6h表達明顯上調(diào);IκBα蛋白于LPS 2h和4h組表達明顯下調(diào);Jmjd3表達亦上調(diào),于1h和2h上調(diào)明顯。(6)染色質(zhì)免疫共沉淀技術(shù)分析目標基因轉(zhuǎn)錄起始結(jié)合位點NF-κB/p65、Jmjd3和H3K27me3的募集關(guān)系:NF-κB/p65關(guān)聯(lián)DNA文庫中IL-6、MMP-9的相對擴增量與相應(yīng)的對照組相比顯著升高(P0.01),而ICAM-1與相應(yīng)對照組相比升高,但差異無統(tǒng)計學意義(P0.05);Jmjd3關(guān)聯(lián)DNA文庫、H3K27me3關(guān)聯(lián)DNA文庫中目標基因擴增結(jié)果與NF-κB/p65關(guān)聯(lián)DNA文庫擴增結(jié)果一致。結(jié)論:LPS誘導內(nèi)皮細胞中NF-κB-Jmjd3信號通路激活,活化高峰時間為2h:NF-κB信號通路活化后釋放轉(zhuǎn)錄因子入核結(jié)合到Jmjd3相應(yīng)啟動子區(qū)域,調(diào)控Jmjd3表達,上調(diào)的Jmjd3入核作用于目標基因區(qū)域組蛋白H3K27me3使其去甲基化、改變構(gòu)象,從而暴露目標基因轉(zhuǎn)錄起始序列,NF-κB通路的活化時也調(diào)控轉(zhuǎn)錄因子本身表達,上調(diào)的核因子-κB結(jié)合到暴露的轉(zhuǎn)錄起始序列上調(diào)控目標基因的轉(zhuǎn)錄,引起炎癥介質(zhì)和粘附分子的表達影響內(nèi)皮細胞微環(huán)境,導致炎癥相關(guān)病理過程。
[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.
【學位授予單位】：南昌大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R730.2
[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.
【學位授予單位】：南昌大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R730.2
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