本文關(guān)鍵詞： 肝炎病毒 乙型 準(zhǔn)種 超深焦磷酸測(cè)序 克隆測(cè)序 遺傳異質(zhì)性　出處：《上海交通大學(xué)》2014年博士論文　論文類型：學(xué)位論文

【摘要】：背景與目的：本課題組之前采用克隆測(cè)序（clone-based sequencing，CBS）的方法研究發(fā)現(xiàn)乙型肝炎病毒（hepatitis B virus, HBV）逆轉(zhuǎn)錄酶（reverse transcriptase, RT）區(qū)準(zhǔn)種異質(zhì)性可作為接受核苷類似物治療的患者抗病毒療效的一個(gè)預(yù)測(cè)指標(biāo)。第二代測(cè)序技術(shù)可進(jìn)行皮升（picoliter）規(guī)模的高通量平行反應(yīng)以檢測(cè)單個(gè)DNA分子，無(wú)需克隆這一費(fèi)時(shí)繁瑣的步驟。本研究首先通過(guò)比較超深焦磷酸測(cè)序（ultra-deeppyrosequencing UDPS），一種新的第二代測(cè)序技術(shù)，和CBS兩種方法分析乙型肝炎病毒RT準(zhǔn)種，結(jié)合生物信息學(xué)分析方法描述準(zhǔn)種遺傳異質(zhì)性特征，探討兩種方法分析HBV生物多樣性的表現(xiàn)力差異及其可能的臨床意義。在此基礎(chǔ)上，應(yīng)用高通量UDPS方法檢測(cè)拉米夫定抗病毒治療早期階段HBV RT區(qū)準(zhǔn)種的動(dòng)態(tài)變化，探討病毒準(zhǔn)種因素對(duì)抗病毒治療應(yīng)答的影響。 研究對(duì)象與方法：本研究的第一部分納入了31例未接受過(guò)抗病毒藥物治療的慢性乙型肝炎患者，收集患者血清，抽提HBV基因組DNA，分別用CBS和UDPS方法平行分析RT區(qū)準(zhǔn)種。用生物信息學(xué)方法對(duì)準(zhǔn)種的異質(zhì)性特征進(jìn)行分析，準(zhǔn)種的復(fù)雜度用下述公式計(jì)算（Sn=i(pi ln pi)/lnN）。本研究的第二部分共納入了35例接受拉米夫定治療至少48周的慢性乙型肝炎患者（16例為病毒學(xué)應(yīng)答者，19例為部分病毒學(xué)應(yīng)答者）。根據(jù)治療48周時(shí)HBV DNA載量將患者分為應(yīng)答組（HBVDNA載量低于檢測(cè)下限）和部分應(yīng)答組（HBV DNA載量高于檢測(cè)下限，但較治療基線水平下降1log10IU/mL）。收集治療基線及治療4周時(shí)的血清，抽提HBV基因組DNA，應(yīng)用聚合酶鏈?zhǔn)剑≒CR）反應(yīng)分別擴(kuò)增HBV逆轉(zhuǎn)錄酶區(qū)三個(gè)相互重疊的約400bp的連續(xù)部分，將PCR產(chǎn)物進(jìn)行超深焦磷酸測(cè)序。采用生物信息學(xué)方法分析準(zhǔn)種的異質(zhì)性特征，分析RT區(qū)準(zhǔn)種基線和4周時(shí)的復(fù)雜度和離散度，并進(jìn)一步分析兩組患者的準(zhǔn)種進(jìn)化模式。 結(jié)果：第一部分：UDPS方法獲得的有效準(zhǔn)種株數(shù)遠(yuǎn)大于CBS方法（P0.001），相關(guān)分析表明UDPS和CBS兩種方法的準(zhǔn)種復(fù)雜度在核苷酸水平具有相關(guān)性（P0.05），UDPS方法準(zhǔn)種的復(fù)雜度在核苷酸和氨基酸水平均高于CBS方法（P0.001）。 RT區(qū)變異分布研究發(fā)現(xiàn)，平均每個(gè)樣本UDPS方法能檢測(cè)到16.2±1.4個(gè)氨基酸變異位點(diǎn)，較CBS方法多9.7±1.1個(gè)位點(diǎn)。UDPS方法相關(guān)的進(jìn)化分析較CBS方法顯示了更多的遺傳信息。第二部分：拉米夫定應(yīng)答組RT區(qū)第1段和第2段的準(zhǔn)種復(fù)雜度在治療基線時(shí)高于部分應(yīng)答組（P 0.05），應(yīng)答組RT區(qū)第3段準(zhǔn)種復(fù)雜度在治療4周時(shí)高于部分應(yīng)答組（P 0.05）。應(yīng)答組的各個(gè)區(qū)段準(zhǔn)種離散度相關(guān)參數(shù)（主要包括平均遺傳距離和平均非同義替換數(shù)）在治療基線時(shí)高于部分應(yīng)答組（P 0.05），應(yīng)答組的第1和第3區(qū)段準(zhǔn)種離散度主要參數(shù)（主要包括平均遺傳距離和平均非同義替換數(shù)）在治療4周時(shí)亦高于部分應(yīng)答組（P 0.05）。此外，，應(yīng)答組RT區(qū)第1段的平均遺傳距離（氨基酸水平）和平均非同義替換數(shù)的平均變化值和凈變化值均高于部分應(yīng)答組（P 0.05）。 結(jié)論：在低豐度變異的檢測(cè)和準(zhǔn)種模擬方面，UDPS方法描述準(zhǔn)種異質(zhì)性的表現(xiàn)力較CBS方法更敏感和高效，盡管UDPS方法尚存在不足，但其為HBV準(zhǔn)種研究未來(lái)向臨床推廣應(yīng)用指明了方向。拉米夫定抗病毒治療初始及早期階段時(shí)病毒學(xué)應(yīng)答患者和部分病毒學(xué)應(yīng)答患者HBV RT區(qū)準(zhǔn)種的動(dòng)態(tài)變化不同，治療基線及治療4周內(nèi)HBV RT區(qū)準(zhǔn)種的異質(zhì)性及動(dòng)態(tài)變化與拉米夫定抗病毒治療的療效相關(guān)。
[Abstract]:Background and purpose: by cloning and sequencing the research group before (clone-based sequencing CBS) method to study found that hepatitis B virus (hepatitis B virus HBV (reverse transcriptase) reverse transcriptase, RT) quasispecies heterogeneity can be used as a predictive index of patients receiving antiviral efficacy of nucleoside analogues therapy. Generation sequencing the technology can be second picoliter scale (picoliter) high-throughput parallel reactions to detect single DNA molecules, without cloning this time-consuming tedious steps. Firstly, through the comparison of ultra deep pyrosequencing (ultra-deeppyrosequencing UDPS), a new second generation sequencing technology, and CBS two methods of analysis of hepatitis B virus RT quasispecies, combined with bioinformatics analysis method to describe the quasispecies genetic heterogeneity, two methods of analysis of differences between HBV biodiversity expression and its clinical significance in May. Based on this, we applied high-throughput UDPS to detect the dynamic changes of quasispecies in HBV RT region at the early stage of lamivudine antiviral therapy, and to explore the influence of viral quasispecies on the response to viral therapy.
Subjects and methods: in the first part of the study included 31 cases of chronic hepatitis B patients had not received antiretroviral treatment, collect serum, extracting the genome of HBV DNA, CBS and UDPS respectively with the method of parallel analysis of RT quasispecies. By the methods of bioinformatics alignment heterogeneity for analysis of complex quasi use the following formula to calculate the degree of (Sn=i (PI ln PI) /lnN). The second part of this study included 35 cases of chronic hepatitis B patients treated with lamivudine for at least 48 weeks (16 cases of virological response, 19 cases with partial virological response patients). According to the treatment at 48 weeks HBV DNA the amount of patients will be divided into response group (load of HBVDNA below the detection limit) and partial response group (HBV DNA load is higher than the detection limit, but compared with the baseline 1log10IU/mL). Collected before treatment and after 4 weeks of treatment the serum, extracting the genome of HBV DN A, polymerase chain reaction (PCR) continuous part were amplified by HBV reverse transcriptase region three overlapping about 400bp, the PCR products were ultra deep pyrosequencing. Bioinformatics analysis of quasi heterogeneity for the analysis of RT quasispecies at baseline and after 4 weeks of complexity and from the divergence, and further analysis of the quasi evolutionary models of two groups of patients.
Results: the first part: a number of effective quasi UDPS method obtained more than CBS method (P0.001), correlation analysis showed that UDPS and CBS two methods of quasispecies complexity at the nucleotide level has correlation (P0.05) method, UDPS quasispecies complexity in nucleotide and amino acid levels were higher than the CBS method (P0.001). The RT variant distribution study found that the average sample UDPS method can detect 1.4 amino acid mutations was 16.2, compared with CBS 9.7 + 1.1.UDPS loci related methods of phylogenetic analysis showed that the genetic information is more than the CBS method. The second part: the response to lamivudine group of RT first and second segments of quasispecies the complexity in the baseline is higher than the partial response group (P 0.05), third groups of RT response section quasispecies complexity in the treatment of 4 weeks is higher than the partial response group (P 0.05). Each sector response group of quasispecies dispersion related parameters (mainly Including the average genetic distance and the average number of nonsynonymous substitutions) is higher than that of partial responders at baseline (P 0.05), the first and third section response group quasi discrete degree of main parameters (including the average genetic distance and the average number of nonsynonymous substitutions) in the treatment of 4 weeks before that of response group (P 0.05). In addition, the average genetic response group RT first distance (amino acid level) and the average change of the average value for the number of non synonymous substitutions and net change values were higher than the partial response group (P 0.05).
Conclusion: the detection of low abundance variation and quasi simulation, UDPS method to describe the quasispecies heterogeneity of expression than the CBS method is more sensitive and efficient, while the UDPS method is insufficient, but the HBV quasispecies study to future clinical application direction. Lamivudine antiviral therapy and early initial stage virological response patients and some patients HBV RT virological response zone quasi dynamic changes of different treatment at baseline and within 4 week of treatment HBV RT quasi heterogeneity and dynamic change and the kind of lamivudine therapy.

【學(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R512.62

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中國(guó)期刊全文數(shù)據(jù)庫(kù) 前1條

1 董菁,成軍,王勤環(huán),施雙雙,洪源,皇甫競(jìng)坤,王剛,李莉,斯崇文;乙型肝炎病毒逆轉(zhuǎn)錄酶區(qū)基因序列準(zhǔn)種與變異特點(diǎn)[J];病毒學(xué)報(bào);2001年03期

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