本文關(guān)鍵詞： 乙型肝炎病毒 慢加急性肝衰竭 表觀遺傳 單卵孿生子 DNA甲基化 基因印跡 小分子RNA　出處：《第三軍醫(yī)大學(xué)》2013年博士論文　論文類型：學(xué)位論文

【摘要】：在我國,慢性HBV感染是引起慢加急性肝衰竭(acute-on-chronic liver failure, ACLF)的主要病因。ACLF病情兇險,進展迅速,預(yù)后較差,雖然近年來在ACLF的治療等方面取得了很大的進展,但病死率仍高達50%～60%。這與ACLF發(fā)病機制尚不明確直接相關(guān),故明確其發(fā)病機制對提高ACLF的治療水平具有重要意義。我們前期對感染HBV的單卵孿生子(monozygotic twins, MZ twins)臨床觀察發(fā)現(xiàn),同樣感染HBV,MZ孿生子間表現(xiàn)出完全不同的臨床表型。MZ孿生子相當(dāng)于天然的“克隆人”,共享遺傳背景,排除了DNA序列差異對基因表達的影響,其HBV感染途徑一致,但表現(xiàn)出明顯不同的表型,考慮可能主要是由于表觀遺傳學(xué)修飾機制所致。近年研究發(fā)現(xiàn)DNA甲基化、小分子RNA (microRNAs, miRNAs)和基因印跡等表觀遺傳調(diào)控機制廣泛參與了病毒復(fù)制、炎癥和免疫調(diào)節(jié)等過程。故我們推測表觀遺傳調(diào)控機制在HBV感染后ACLF的發(fā)病中可能起重要作用。 了解DNA甲基化、miRNAs和基因印跡等表觀遺傳調(diào)控機制在ACLF患者與對照樣本之間差異表達有助于篩選出與ACLF相關(guān)的表觀遺傳現(xiàn)象,為進一步闡明表觀遺傳調(diào)控在ACLF發(fā)病中的具體機制奠定基礎(chǔ)。所以我們以HBV感染后表型分別為ACLF和無癥狀攜帶者(asymptomatic carrier, AsC)的一對MZ孿生子標(biāo)本為基礎(chǔ),采用以下方法觀察了ACLF和AsC患者間DNA甲基化、基因印跡和miRNAs表達的差異。以期了解表觀遺傳調(diào)控與ACLF的關(guān)系。 方法： 1.采用Agilent Human CpG Islands arrays芯片對MZ孿生子進行了全基因組差異甲基化芯片掃描,獲得HBV感染后表型不同MZ孿生子間差異甲基化基因譜。通過Roche Nimblegen表達譜芯片進行全基因組表達掃描,獲得HBV感染后表型不同MZ孿生子間差異表達基因譜。聯(lián)合分析甲基化基因的表達和基因已知功能篩選出可能與ACLF發(fā)病相關(guān)的甲基化基因。 2.通過表達譜芯片結(jié)果篩選出MZ孿生子間差異表達印跡基因,并采用熒光定量RT-PCR在52例HBV感染后ACLF患者和48例AsC患者間驗證差異表達情況。然后采用PCR—限制性片斷長度多態(tài)性(restriction fragment length polymorphism, RFLP)分析篩選IGF2基因雜合標(biāo)本；并用基于RT-PCR的RFLP分析檢測雜合標(biāo)本的印跡狀態(tài)。采用χ2檢驗分析印跡基因IGF2在ACLF患者和AsC患者印跡狀態(tài)差異。 3.采用Exiqon miRNA Array芯片完成孿生子間差異1niRNA表達譜掃描,獲得差異表達miRNA譜。并采用熒光定量RT-PCR在104例HBV感染后ACLF患者和96例AsC患者間驗證表達差異。Targetscan、iRanda和PicTar數(shù)據(jù)庫多重比對等方法預(yù)測差異表達miRNA靶基因。 主要研究結(jié)果： 1.在ACLF MZ孿生子有47條基因特異性甲基化且表達下調(diào)或無表達；有88條基因特異性非甲基化且表達上調(diào),其中28條基因僅在ACLF MZ孿生子有表達。這些差異表達基因與炎癥信號通路,炎癥介質(zhì)表達,微循環(huán)障礙等密切相關(guān)。生物信息學(xué)分析發(fā)現(xiàn)以下7條基因的異常甲基化可能與肝炎發(fā)病相關(guān)：ZEB1、USP47、BTF3L4、 ADCYAP1、 PLCH2、F10和PAK6。 2.發(fā)現(xiàn)ACLF MZ孿生子中上調(diào)印跡基因13條：DDC、TFPI2、KLF14、ABCA1、 IGF2、IGF2AS、KCNQ1DN、CDKN1C、SLC22A18AS、MEG3、DLK1、 ATP10A及TCEB3C;下調(diào)印跡基因8條：SGCE、PEG10、DLX5、SNRPN、MKRN3、MAGEL2. NNAT及L3MBTL2�？赡芘c肝臟炎癥相關(guān)的IGF2、DKN1C和TFPI2均在ACLF患者表達增高(P0.001)。IGF2在ACLF患者發(fā)生基因印跡缺失(loss of imprinting, LOI)較AsC患者明顯增加(55.56%vs.21.05%,P=0.045)。 3.在MZ孿生子間發(fā)現(xiàn)差異表達miRNA53條；45條在ACLF MZ表達上調(diào),8條表達下調(diào)。hsa-let-7a、hsa-mir-16在ACLF患者表達明顯增高,分別增高8.58和8.63倍(P0.001)。靶基因預(yù)測結(jié)果發(fā)現(xiàn)BCL2、CARD8、EDA、IL1RAPL1、LTB及FZD10可能為hsa-mir-16參與ACLF發(fā)病的靶基因；CERCAM、IGF2BP1、OPRM1及MAP4K3可能為hsa-let-7a參與ACLF發(fā)病的靶基因。 結(jié)論： 1.HBV感染后ACLF患者與AsC患者間存在基因差異甲基化、印跡基因和miRNAs表達差異等表觀遺傳漂移現(xiàn)象。證實表觀遺傳調(diào)控可能參與了ACLF的發(fā)生。 2.IGF2在ACLF患者表達較AsC患者明顯增加,其原因與IGF2在ACLF患者發(fā)生基因印跡缺失的比例增加有關(guān)。 3.let-7a、mir-16在ACLF患者表達明顯增高。mir-16可能通過調(diào)節(jié)BCL2、ARD8等的表達；let-7a可能通過調(diào)節(jié)IGF2BP1、MAP4K3等的表達參與肝臟炎癥的發(fā)生。
[Abstract]:In our country, chronic HBV infection is caused by acute on chronic liver failure (acute-on-chronic liver failure, ACLF.ACLF) the main cause of serious disease, rapid progression, poor prognosis, although in recent years in the treatment of ACLF made great progress, but the mortality rate is still as high as 50% ~ 60%. and the pathogenesis of ACLF is not so clear is directly related to clarify its pathogenesis has important significance to improve the treatment level of ACLF. Our previous child of HBV infected monozygotic twins (monozygotic twins, MZ twins) clinical observation found that the same HBV infection, MZ twins showed different clinical phenotypes of.MZ twins is equivalent to the natural "human cloning, shared genetic background, excluding the impact of DNA sequence differences in gene expression, the same way the HBV infection, but showed significantly different phenotypes, consideration is likely due to epigenetic Due to recent studies indicate that DNA methylation, small molecule RNA (microRNAs, miRNAs) and gene imprinting mechanisms of epigenetic regulation is involved in a wide range of viral replication, inflammation and immune regulation process. So we speculate that the incidence table epigenetic regulation mechanism of ACLF in HBV after infection could play an important role.
Understanding of DNA methylation, miRNAs and gene imprinting mechanisms of epigenetic regulation in the sample differences between ACLF patients and control the expression of help in the selection of ACLF related epigenetic phenomena, lay the foundation for further understanding the specific mechanism of epigenetic regulation in the pathogenesis of ACLF. So we use HBV phenotype after infection respectively ACLF and asymptomatic carriers (asymptomatic carrier, AsC MZ) a pair of twins were observed based on ACLF and AsC between patients with DNA methylation by using the following methods differentially expressed gene imprinting and miRNAs. In order to understand the relationship between epigenetic regulation and ACLF.
Method錛，

本文編號：1533768