本文選題：基因芯片 + lncRNA　； 參考：《南方醫(yī)科大學》2017年碩士論文

【摘要】：研究背景結(jié)核分枝桿菌(Mycobacterium tuberculosis,MTB)是引起結(jié)核病的致病菌。MTB能通過多種免疫逃逸機制逃避巨噬細胞(macrophage,Mφ)的清除,從而長期寄生在Mφ內(nèi)。長鏈非編碼RNA(long noncoding RNA,lncRNA)廣泛參與多種生物學進程并發(fā)揮關(guān)鍵作用,且可以作為多種疾病的診斷標志物和潛在的治療靶標。但是lncRNA能否作為結(jié)核病的診斷標志物以及在MTB感染Mφ中的作用仍未清楚。研究目的通過基因芯片檢測MTB感染Mφ后lncRNA和mRNA表達譜的變化,篩選有望用于結(jié)核病診斷的生物標志物;初步探討了lncRNA—ENST00000360485在MTB感染Mφ中的作用,以期發(fā)現(xiàn)Mφ抵抗MTB感染的新機制。研究方法分別用MTB弱毒株H37Ra和強毒株H37Rv感染人原代巨噬細胞,使用lncRNA芯片(Arraystar Human LncRNA Microarray V3.0)檢測被感染巨噬細胞lncRNA和mRNA表達譜,以未感染Mφ為對照;分別挑選6條lncRNA和6條mRNA,使用實時熒光定量PCR(quantitative real-time PCR,qRT-PCR)技術(shù)驗證基因芯片結(jié)果的可重復(fù)性;使用生物信息學方法對差異表達的mRNA進行GO分析(Gene Ontology analysis,GO analysis)和 KEGG 通路分析(Kyoto Encyclopedia of Genes and Genomes(KEGG)biological pathway analysis);使用qRT-PCR檢測32例健康志愿者和31例結(jié)核病患者的PBMC樣本,篩選有望用于結(jié)核病診斷的生物標志物;使用siRNA抑制Mφ的lncRNA-ENST00000360485表達后,感染BCG,qRT-PCR檢測其鄰近基因和Mφ細胞因子TNF-α、IL-1β和IL-6的RNA表達水平變化,采用Western Blot檢測Mφ自噬水平的變化,通過克隆形成實驗(colony-forming unit,CFU)檢測Mφ清除MTB的效果。結(jié)果基因芯片結(jié)果顯示,H37Ra感染后,Mφ有972條lncRNA和2138條mRNA出現(xiàn)差異性表達,而H37Rv感染后,Mφ有1417條lncRNA和2478條mRNA出現(xiàn)差異性表達;所挑選的lncRNA和mRNA的表達趨勢與基因芯片結(jié)果高度一致;GO和KEGG通路分析結(jié)果分別顯示了 H37Ra和H37Rv感染相關(guān)的功能和代謝通路;qRT-PCR檢測發(fā)現(xiàn)3個在健康對照和結(jié)核病人之間存在顯著性差異表達的lncRNA(ENST00000360485,MIR3945HG V1 和MIR3945HG V2);ROC曲線分析結(jié)果顯示:ENST00000360485、MIR3945HG V1、MIR3945HG V2的ROC曲線下面積(AUC)為0.798、0.925、0.956,敏感性分別是83.97%、90%、89.66%,特異性分別是71.88%、81.25%、90.63%;siRNA抑制ENST00000360485表達前后,ENST00000360485的上下游鄰近基因(RAP1B和MDM1)和Mφ細胞因子TNF-α、IL-1β和IL-6的RNA表達水平?jīng)]有變化,Mφ自噬通路的活化水平和清除MTB的能力也沒有顯著性差異。結(jié)論本研究發(fā)現(xiàn),H37Rv和H37Ra誘導(dǎo)Mφ的lncRNA和mRNA表達譜具有顯著性差異;生物信息學分析了差異表達的mRNA所富集的功能和代謝通路的差異;初步驗證了3個lncRNA--ENST00000360485、MIR3945HG V1和MIR3945HG V2作為結(jié)核病診斷標志物的可行性;并對ENST00000360485功能的初步探討,為后續(xù)深入研究奠定了基礎(chǔ)。
[Abstract]:Background Mycobacterium tuberculosism MTB (Mycobacterium tuberculosisus MTB) is a pathogenic bacterium causing tuberculosis. MTB can escape the clearance of macrophage macrophage M 蠁 through a variety of immune escape mechanisms, so that it can be parasitized in M 蠁 for a long time.Long-chain noncoding RNA(long noncoding RNAs play a key role in many biological processes, and can be used as diagnostic markers and potential therapeutic targets for many diseases.However, whether lncRNA can be used as a diagnostic marker of tuberculosis and its role in M 蠁 infection of MTB remains unclear.Objective to detect the changes of lncRNA and mRNA expression profiles after MTB infection with M 蠁 by gene microarray, and to screen biomarkers that could be used in the diagnosis of tuberculosis, and to explore the role of lncRNA-ENST00000360485 in M 蠁 infection with MTB.The aim of this study was to find a new mechanism of M 蠁 resistance to MTB infection.Methods Human primary macrophages were infected with MTB attenuated H37Ra and virulent H37Rv, respectively. The lncRNA and mRNA expression profiles of infected macrophages were detected by lncRNA Human Human LncRNA Microarray V3.0.Six lncRNA and six mRNAs were selected, and the reproducibility of the results was verified by real-time fluorescence quantitative PCR(quantitative real-time real-time qRT-PCRR.Using bioinformatics method, go analysis of differentially expressed mRNA (go analysis) and KEGG pathway analysis of Genes and Genomes(KEGG)biological pathway analysis were performed. PBMC samples of 32 healthy volunteers and 31 tuberculosis patients were detected by qRT-PCR.After siRNA was used to inhibit the expression of M 蠁 lncRNA-ENST00000360485, the expression of adjacent genes and M 蠁 cytokines, TNF- 偽, IL-1 尾 and IL-6 were detected by RT-PCR, and M 蠁 autophagy was detected by Western Blot.Colony forming assay (CFU) was used to detect the MTB scavenging effect of M 蠁.Results there were 972 lncRNA and 2138 mRNA differentially expressed in M 蠁 after H37Ra infection, and 1417 lncRNA and 2478 mRNA in M 蠁 after H37Rv infection.The expression trend of selected lncRNA and mRNA was highly consistent with the results of gene chip analysis. The results of go and KEGG pathway analysis showed that H37Ra and H37Rv infection related function and metabolic pathway were detected by qRT-PCR in 3 healthy controls and TB patients, respectively.涔嬮棿瀛樺湪鏄捐憲鎬у樊寮傝〃杈劇殑lncRNA(ENST00000360485,MIR3945HG V1 鍜孧IR3945HG V2);ROC鏇茬嚎鍒嗘瀽緇撴灉鏄劇ず:ENST00000360485,MIR3945HG V1,MIR3945HG V2鐨凴OC鏇茬嚎涓嬮潰縐，

本文編號：1741828