本文選題：EB病毒 + 立即早期蛋白Rta　； 參考：《南京醫(yī)科大學》2014年博士論文

【摘要】：EB病毒感染在兒童期多表現(xiàn)為傳染性單核細胞增多癥(Infectious mononucleosis, IM),部分可演變?yōu)閲乐氐氖妊毎C合癥及淋巴瘤。干擾素調(diào)節(jié)因子3(interferon regulatory factor3, IRF-3)是調(diào)節(jié)I型干擾素(IFN-a/p)基因表達的關(guān)鍵轉(zhuǎn)錄因子,在固有免疫反應(yīng)和獲得性免疫反應(yīng)中起重要作用。但是EB病毒調(diào)控機體干擾素表達以逃避宿主免疫的具體機制尚不十分清楚。 本研究通過Real time PCR法檢測臨床EB病毒感染的IM病人與健康對照組相關(guān)因子差異表達,發(fā)現(xiàn)IM病人中立即早期蛋白復(fù)制與轉(zhuǎn)錄激活子(replication and transcription activator, Rta)、miRNA146a、miRNA23a、miRNA24表達顯著高于健康對照組,而IRF-3及IFNβ表達顯著低于健康對照組。為探索Rta與miRNA是否調(diào)控IRF-3及IFNβ表達及相關(guān)機制,我們將Rta過表達質(zhì)粒與含IRF-3啟動子區(qū)不同片段的螢光素酶報告質(zhì)粒共轉(zhuǎn)染于HeLa細胞,發(fā)現(xiàn)Rta對含-149至-93bp片段的IRF-3啟動子重組質(zhì)粒螢光素酶活性影響較顯著。既往研究報道,轉(zhuǎn)錄因子E2F1可與IRF-3啟動子-149/-93bp區(qū)域內(nèi)E2F1位點結(jié)合,抑制IRF-3轉(zhuǎn)錄表達。本研究通過E2F1結(jié)合位點點突變、過表達E2F1、RNA干擾E2F1表達及染色質(zhì)免疫沉淀實驗證實,在HeLa細胞中Rta可通過上調(diào)E2F1表達、增加其與IRF-3啟動子區(qū)的結(jié)合下調(diào)IRF-3的表達。通過miRNA過表達及干擾,發(fā)現(xiàn)miRNA146a、miRNA23a、miRNA24可下調(diào)IRF-3mRNA表達,而miRNA146a可下調(diào)IRF-3蛋白表達。我們分別將miRNA146a、 miRNA23a、miRNA24與IRF-3啟動子重組螢光素酶報告質(zhì)粒共轉(zhuǎn)染,發(fā)現(xiàn)啟動子活性無顯著降低,提示上述miRNA并非通過啟動子區(qū)發(fā)揮負性調(diào)控作用。進一步實驗發(fā)現(xiàn)miRNA146a可下調(diào)IFNβ信號通路上游分子TRAF6、IRAK1、 NF-κB等表達。通過TRAF6過表達及干擾實驗證實,TRAF6在miRNA146a調(diào)控IFNβ表達中起重要介導(dǎo)作用。丙戊酸(Valproic acid, VPA)作為組蛋白去乙酰化酶抑制劑,不同細胞中均可上調(diào)IRF-3mRNA及蛋白表達,深入研究發(fā)現(xiàn)丙戊酸可通過增加組蛋白H3、H4的乙�；龠MIRF-3轉(zhuǎn)錄,增加IRF-3基因表達,而免疫熒光實驗進一步證實丙戊酸可明顯增加293T細胞核磷酸化IRF-3的表達。 本研究發(fā)現(xiàn)：Rta可通過上調(diào)E2F1表達、增加其與IRF-3啟動子區(qū)的結(jié)合從而抑制IRF-3的表達；miRNA146a通過TRAF6抑制IFNβ轉(zhuǎn)錄表達；丙戊酸可通過增加組蛋白乙�；龠MIRF-3的轉(zhuǎn)錄表達及磷酸化。本研究拓展了EB病毒逃避宿主免疫機制的探索,完善了EB病毒誘導(dǎo)Rta及miRNA調(diào)控IRF-3的分子機制,且為今后臨床治療EB病毒相關(guān)疾病提供實驗依據(jù)。
[Abstract]:Epstein-Barr virus (EBV) infection in childhood is characterized by infectious mononucleosis (IM), which can develop into severe haemophilic syndrome and lymphoma. Interferon regulatory factor 3 (interferon regulatory factor3 (IRF-3) is a key transcription factor that regulates the expression of IFN-ap gene and plays an important role in innate and acquired immune responses. However, the specific mechanism of EB virus regulating interferon expression to escape host immunity is unclear. In this study, the differential expression of related factors in IM patients infected with Epstein-Barr virus (EBV) and healthy controls was detected by Real time PCR. It was found that the expression of miRNA146ahmiRNA23aRNA24 in IM patients was significantly higher than that in healthy controls, and the expression of immediate early protein replication and activator of transcription (replication and transcription activator, Rta) in IM patients was significantly higher than that in healthy controls. The expression of IRF-3 and IFN- 尾 was significantly lower than that of healthy control group. In order to investigate whether Rta and miRNA can regulate the expression of IRF-3 and IFN- 尾, we co-transfected RTA overexpression plasmid and luciferase reporter plasmid containing different fragments of IRF-3 promoter into HeLa cells. It was found that RTA had a significant effect on the luciferase activity of the recombinant plasmid of IRF-3 promoter containing -149 to -93 BP fragments. It has been reported that transcription factor E2F1 can bind to the E2F1 site of IRF-3 promoter -149 / 93bp and inhibit the expression of IRF-3 transcription. In this study, the overexpression of E2F1RNA interfering with E2F1 expression and chromatin immunoprecipitation assay demonstrated that Rta could up-regulate the expression of E2F1 and down-regulate the expression of IRF-3 by up-regulating the expression of E2F1 in HeLa cells. By over-expression and interference of miRNA, it was found that miRNA146a miRNA23a miRNA24 could down-regulate the expression of IRF-3 mRNA, while miRNA146a could down-regulate the expression of IRF-3 protein. We cotransfected the recombinant luciferase reporter plasmids of miRNA146a, miRNA23ahmiRNA24 and IRF-3 promoter, and found that the promoter activity was not significantly decreased, suggesting that the miRNA does not play a negative regulatory role through the promoter region. It was further found that miRNA146a could down-regulate the expression of TRAF6- IRAK1 and NF- 魏 B upstream molecules in IFN- 尾 signaling pathway. The overexpression of TRAF6 and interference experiments confirmed that TRAF6 plays an important role in the regulation of IFN- 尾 expression by miRNA146a. Valproic acid (VPA), as a histone deacetylase inhibitor, can up-regulate the expression of IRF-3 mRNA and protein in different cells. It has been found that valproic acid can promote the transcription of IRF-3 and increase the expression of IRF-3 by increasing the acetylation of histone H3H4. Immunofluorescence assay further confirmed that valproic acid could significantly increase the expression of phosphorylated IRF-3 in 293T nucleus. In this study, we found that: Rta could inhibit the expression of IRF-3 by up-regulating the expression of E2F1, increasing its binding to IRF-3 promoter region and inhibiting the transcription of IFN- 尾 by TRAF6, and valproic acid could promote the transcription and phosphorylation of IRF-3 by increasing histone acetylation. This study expanded the mechanism of EBV evading host immunity, improved the molecular mechanism of EBV induced Rta and miRNA regulating IRF-3, and provided experimental evidence for clinical treatment of EBV related diseases.
【學位授予單位】：南京醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R725.1

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相關(guān)期刊論文 前1條

1 謝正德;申昆玲;;重視兒童非腫瘤性EB病毒感染疾病的研究[J];首都醫(yī)科大學學報;2010年02期

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本文編號：2099565