本文選題：EB病毒 + 立即早期蛋白R(shí)ta　； 參考：《南京醫(yī)科大學(xué)》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(xì)胞增多癥(Infectious mononucleosis, IM),部分可演變?yōu)閲?yán)重的嗜血細(xì)胞綜合癥及淋巴瘤。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(dá)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(jī)體干擾素表達(dá)以逃避宿主免疫的具體機(jī)制尚不十分清楚。 本研究通過Real time PCR法檢測(cè)臨床EB病毒感染的IM病人與健康對(duì)照組相關(guān)因子差異表達(dá),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(dá)顯著高于健康對(duì)照組,而IRF-3及IFNβ表達(dá)顯著低于健康對(duì)照組。為探索Rta與miRNA是否調(diào)控IRF-3及IFNβ表達(dá)及相關(guān)機(jī)制,我們將Rta過表達(dá)質(zhì)粒與含IRF-3啟動(dòng)子區(qū)不同片段的螢光素酶報(bào)告質(zhì)粒共轉(zhuǎn)染于HeLa細(xì)胞,發(fā)現(xiàn)Rta對(duì)含-149至-93bp片段的IRF-3啟動(dòng)子重組質(zhì)粒螢光素酶活性影響較顯著。既往研究報(bào)道,轉(zhuǎn)錄因子E2F1可與IRF-3啟動(dòng)子-149/-93bp區(qū)域內(nèi)E2F1位點(diǎn)結(jié)合,抑制IRF-3轉(zhuǎn)錄表達(dá)。本研究通過E2F1結(jié)合位點(diǎn)點(diǎn)突變、過表達(dá)E2F1、RNA干擾E2F1表達(dá)及染色質(zhì)免疫沉淀實(shí)驗(yàn)證實(shí),在HeLa細(xì)胞中Rta可通過上調(diào)E2F1表達(dá)、增加其與IRF-3啟動(dòng)子區(qū)的結(jié)合下調(diào)IRF-3的表達(dá)。通過miRNA過表達(dá)及干擾,發(fā)現(xiàn)miRNA146a、miRNA23a、miRNA24可下調(diào)IRF-3mRNA表達(dá),而miRNA146a可下調(diào)IRF-3蛋白表達(dá)。我們分別將miRNA146a、 miRNA23a、miRNA24與IRF-3啟動(dòng)子重組螢光素酶報(bào)告質(zhì)粒共轉(zhuǎn)染,發(fā)現(xiàn)啟動(dòng)子活性無顯著降低,提示上述miRNA并非通過啟動(dòng)子區(qū)發(fā)揮負(fù)性調(diào)控作用。進(jìn)一步實(shí)驗(yàn)發(fā)現(xiàn)miRNA146a可下調(diào)IFNβ信號(hào)通路上游分子TRAF6、IRAK1、 NF-κB等表達(dá)。通過TRAF6過表達(dá)及干擾實(shí)驗(yàn)證實(shí),TRAF6在miRNA146a調(diào)控IFNβ表達(dá)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(xì)胞中均可上調(diào)IRF-3mRNA及蛋白表達(dá),深入研究發(fā)現(xiàn)丙戊酸可通過增加組蛋白H3、H4的乙�；龠M(jìn)IRF-3轉(zhuǎn)錄,增加IRF-3基因表達(dá),而免疫熒光實(shí)驗(yàn)進(jìn)一步證實(shí)丙戊酸可明顯增加293T細(xì)胞核磷酸化IRF-3的表達(dá)。 本研究發(fā)現(xiàn)：Rta可通過上調(diào)E2F1表達(dá)、增加其與IRF-3啟動(dòng)子區(qū)的結(jié)合從而抑制IRF-3的表達(dá)；miRNA146a通過TRAF6抑制IFNβ轉(zhuǎn)錄表達(dá)；丙戊酸可通過增加組蛋白乙�；龠M(jìn)IRF-3的轉(zhuǎn)錄表達(dá)及磷酸化。本研究拓展了EB病毒逃避宿主免疫機(jī)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(jī)制,且為今后臨床治療EB病毒相關(guān)疾病提供實(shí)驗(yàn)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(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R725.1

【參考文獻(xiàn)】

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(xué)學(xué)報(bào);2010年02期

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本文編號(hào)：2099565