本文關(guān)鍵詞： HBV整合 共價(jià)閉合環(huán)狀DNA 高通量測(cè)序 適時(shí)定量聚合酶鏈反應(yīng)　出處：《武漢大學(xué)》2014年博士論文　論文類型：學(xué)位論文

【摘要】：乙型肝炎病毒(HBV)感染是嚴(yán)重威脅人民生命健康的疾病。乙肝感染肝炎遷移不愈、并可能發(fā)展為肝細(xì)胞癌(HCC)的根源在于肝細(xì)胞核內(nèi)cccDNA池的持續(xù)存在及HBV整合入宿主細(xì)胞。我們采用改進(jìn)后的靈敏、特異、簡(jiǎn)便的SYBR Green I RT-PCI(?)法及高通量序列捕獲測(cè)序技術(shù)分別對(duì)急性自限性乙肝患者(AHB)、抗病毒治療不同預(yù)后慢性乙肝患者(CHB)的肝細(xì)胞cccDNA水平及HBV整合情況進(jìn)行檢測(cè),確定可代表CHB治愈的肝細(xì)胞cccDNA閾值,了解乙肝患者HBV整合的一般情況。 第一部分：cccDNA檢測(cè)方法學(xué)的建立 目的：建立靈敏、特異、簡(jiǎn)便的肝細(xì)胞HBV cccDNA檢測(cè)方法。 方法：從PSAD酶切、熒光采集溫度、肝組織DNA提取等方面對(duì)傳統(tǒng)cccDNA SYBR Green I RT-PCR檢測(cè)法進(jìn)行改進(jìn)。 結(jié)果：改進(jìn)后的cccDNASYBR Green I RT-PCR檢測(cè)方法簡(jiǎn)便,特異性好,靈敏度高,其檢測(cè)下限值達(dá)24copies/μl。 結(jié)論;簡(jiǎn)便、靈敏、特異的肝細(xì)胞cccDNA檢測(cè)方法的建立為臨床大量乙肝患者肝細(xì)胞cccDNA檢測(cè)奠定了基礎(chǔ)。 第二部分：肝細(xì)胞內(nèi)cccDNA水平對(duì)抗病毒治療CHB患者預(yù)后的預(yù)測(cè)價(jià)值研究 目的：探討肝細(xì)胞內(nèi)cccDNA水平對(duì)抗病毒治療CHB患者預(yù)后的預(yù)測(cè)價(jià)值及與患者病毒學(xué)、血清學(xué)、病理指標(biāo)的相關(guān)性。 方法：對(duì)60名乙肝患者,包括11名AHB患者、46名接受抗病毒治療CHB患者[21名原發(fā)性治療失敗、11名實(shí)現(xiàn)持續(xù)病毒學(xué)反應(yīng)(VR)、15名實(shí)現(xiàn)持續(xù)VR和HBsAg血清學(xué)清除]及3名自身免疫性肝炎患者的肝細(xì)胞cccDNA和總DNA(tDNA)水平、血清HBV DNA、HBsAg、HBeAg和ALT水平進(jìn)行檢測(cè)。 結(jié)果：AHB患者肝細(xì)胞cccDNA和tDNA水平(分別為0.002copies/cell和0.04copies/cell)顯著低于CHB患者。實(shí)現(xiàn)持續(xù)性VR和HBsAg血清學(xué)清除CHB患者的肝細(xì)胞cccDNA水平(0.012copies/cell)顯著低于原發(fā)性治療失敗患者(4.18copies/cell, P㩳0.0001),但和實(shí)現(xiàn)持續(xù)性VR患者無(wú)顯著差異(0.039copies/cell,闡.169)。實(shí)現(xiàn)持續(xù)性VR和HBsAg血清學(xué)清除CHB患者的肝細(xì)胞平均tDNA水平(0.096copies/cell)顯著低于原發(fā)性治療失敗患者(371copies/cell, P㩳0.0001)及實(shí)現(xiàn)持續(xù)性VR患者(1.62copies/cell, P=0.001)。 ROC曲線分析顯示肝細(xì)胞cccDNA水平(0.015copies/cell)和tDNA水平(0.23copies/cell)在預(yù)測(cè)實(shí)現(xiàn)持續(xù)性VR和HBsAg血清學(xué)清除方面沒(méi)有顯著差異(ROC曲線下面積分別為0.88和0.96,P0.10)。肝細(xì)胞cccDNA水平和血清ALT、HBeAg及肝細(xì)胞tDNA水平顯著正相關(guān)(P=0.024,P=0.001和P0.0001),和血清HBV DNA無(wú)相關(guān)性(P=0.12),和血清HBeAg陰性及及HBeAg陽(yáng)性患者血清HBsAg水平無(wú)相關(guān)性(P=0.84和P-0.146)。 結(jié)論：AHB和抗病毒治療后實(shí)現(xiàn)持續(xù)性VR和HBsAg血清學(xué)清除的CHB患者肝細(xì)胞內(nèi)存有極微量的cccDNA。肝細(xì)胞cccDNA水平檢測(cè)有希望成為預(yù)測(cè)抗病毒治療預(yù)后的可靠指標(biāo)。 第三部分：高通量測(cè)序及Sanger測(cè)序檢測(cè)乙肝患者HBV整合研究 目的：探討乙肝攜帶者、AHB及抗病毒治療CHB患者的HBV整合情況。 方法：采用高通量序列捕獲測(cè)序技術(shù)檢測(cè)2名乙肝攜帶者、3名AHB患者及13名抗病毒治療后CHB患者HBV整合狀況。對(duì)整合位點(diǎn)reads支持大于2個(gè)的14個(gè)整合位點(diǎn)用常規(guī)PCR和Sanger測(cè)序進(jìn)行驗(yàn)證,并檢測(cè)這14個(gè)整合位點(diǎn)在全部18名乙肝患者中的存在情況。 結(jié)果：高通量測(cè)序結(jié)果顯示18名乙肝患者中HBV整合陽(yáng)性率為100%,患者整合位點(diǎn)總數(shù)為2083個(gè),平均整合位點(diǎn)為138.2±379.9個(gè)/人,其中原發(fā)性失敗患者HBV平均整合位點(diǎn)數(shù)目最多(248.5±57.3個(gè)/人),實(shí)現(xiàn)持續(xù)性VR患者次之(18.6±13.7個(gè)/人)。乙肝患者HBV整合位點(diǎn)數(shù)目與肝細(xì)胞cccDNA水平、平均覆蓋度及肝細(xì)胞HBsAg積分顯著正相關(guān)(均為P0.0001)。Sanger測(cè)序法驗(yàn)證14個(gè)整合位點(diǎn)成功率為100%,18名乙肝患者均出現(xiàn)整合位點(diǎn)Chr16：51320015。 結(jié)論：乙肝患者HBV整合陽(yáng)性率為100%,其整合位點(diǎn)數(shù)目和患者檢測(cè)平均覆蓋度、肝細(xì)胞HBsAg積分顯著正相關(guān)。整合位點(diǎn)Chr16：51320015為廣泛發(fā)生的主要整合位點(diǎn)(MIS)。 第四部分：HBV整合病毒-宿主基因嵌合體轉(zhuǎn)錄表達(dá)和整合位點(diǎn)Chr16：51320015定量檢測(cè)研究 目的：研究14個(gè)HBV整合位點(diǎn)的病毒-宿主基因嵌合體轉(zhuǎn)錄表達(dá),并對(duì)整合位點(diǎn)Chr16：51320015進(jìn)行定量檢測(cè)。 方法：采用反轉(zhuǎn)錄PCR法檢測(cè)14個(gè)HBV整合位點(diǎn)的病毒-宿主基因嵌合體轉(zhuǎn)錄表達(dá),采用分子克隆及熒光定量PCR法定量檢測(cè)18例乙肝患者的整合位點(diǎn)Chr16:51320015拷貝數(shù)。 結(jié)果：嵌合體轉(zhuǎn)錄表達(dá)研究顯示1、3、4、5、6、12、14號(hào)整合位點(diǎn)出現(xiàn)病毒-宿主基因嵌合體轉(zhuǎn)錄表達(dá),其中除4號(hào)HBV整合區(qū)域?yàn)镾區(qū)外、6號(hào)為前C區(qū)外,其余均為X區(qū)。18例患者整合位點(diǎn)Chr16：51320015均值為1.46×10-2±4.94×10-2copies/cell,其中最大值為0.212copies/cell,最小值為3.48x1O-5copies/cell。 結(jié)論：病毒-宿主基因轉(zhuǎn)錄表達(dá)與HBx基因整合密切相關(guān),HBsAg可以由整合S區(qū)復(fù)制產(chǎn)生。18例Chr16：51320015整合位點(diǎn)均為高拷貝克隆擴(kuò)增。
[Abstract]:Hepatitis B virus (HBV) infection is a serious threat to people's life and health disease. The infection of HBV hepatitis transfer does not heal, and may develop into liver cell carcinoma (HCC) is the source of persistent cccDNA cell nuclei of hepatocytes and HBV integration into the host cell. We use the improved sensitive, specific, simple SYBR Green I RT-PCI (?) and high-throughput sequencing of acute self limited hepatitis B patients (AHB), antiviral therapy with different prognosis in patients with chronic hepatitis B (CHB) were detected in liver cells cccDNA and HBV integration, can determine the hepatocyte cccDNA threshold represents the CHB cure, general understanding of HBV in patients with hepatitis B the integration.
The first part: the establishment of cccDNA detection methodology
Objective: to establish a sensitive, specific and simple method for the detection of HBV cccDNA in liver cells.
Methods: the traditional cccDNA SYBR Green I RT-PCR detection method was improved from the aspects of PSAD enzyme cutting, fluorescence collecting temperature and DNA extraction of liver tissue.
Results: the improved cccDNASYBR Green I RT-PCR detection method is simple, good specificity, high sensitivity, and its detection limit value is 24copies/ Mu L.
Conclusion: the establishment of a simple, sensitive and specific method for the detection of hepatocyte cccDNA has laid a foundation for the detection of hepatocyte cccDNA in patients with large number of hepatitis B patients.
Second part: the predictive value of intrahepatic cccDNA level against the prognosis of CHB patients treated with virus
Objective: To explore the predictive value of cccDNA level in hepatocytes against the prognosis of CHB patients and the correlation with patients' virology, serology, and pathological indexes.
Methods: 60 patients with hepatitis B, including 11 AHB patients, 46 received antiviral therapy in patients with [21 CHB primary treatment failure, 11 patients achieved sustained virological response (VR), VR and HBsAg 15 to achieve sustained clearance] and 3 serological autoimmune hepatitis liver cells cccDNA and DNA (tDNA) the level of serum HBV, DNA, HBsAg, HBeAg and ALT levels were detected.
Results: AHB liver cells in patients with cccDNA and tDNA levels (0.002copies/cell and 0.04copies/cell) was significantly lower than that of CHB patients. To achieve sustained VR and HBsAg serological elimination of liver cccDNA levels in patients with CHB (0.012copies/cell) was significantly lower than that of patients with primary treatment failure (4.18copies/cell, P? 0.0001), but realizing the persistent VR with no significant differences (0.039copies/cell,.169). To achieve sustained VR and HBsAg serological elimination of liver average level of tDNA in patients with CHB (0.096copies/cell) was significantly lower than that of patients with primary treatment failure (371copies/cell, P? 0.0001) and patients with persistent VR (1.62copies/cell, P=0.001). ROC curve analysis showed that the liver cells (cccDNA 0.015copies/cell) and the level of tDNA (0.23copies/cell) in predicting the implementation of persistent VR and HBsAg serological clearance no remarkable difference (area under the ROC curve were 0. 88 and 0.96, P0.10). The cccDNA level in liver cells was positively correlated with serum ALT, HBeAg and tDNA level (P=0.024, P=0.001 and P0.0001). There was no correlation between serum cccDNA level and serum HBV DNA (P=0.12). There was no correlation between serum cccDNA level and serum HBV level.
Conclusion: after detecting AHB and antiviral therapy, the detection of cccDNA level in hepatocytes of patients with persistent VR and HBsAg serological scavenging is very promising in predicting the prognosis of CHB.
Third part: high throughput sequencing and Sanger sequencing for detection of HBV integration in hepatitis B patients
Objective: To investigate the HBV integration of hepatitis B carriers, AHB and CHB patients with antiviral therapy.
Methods: using high-throughput sequencing to detect 2 hepatitis B carriers, the integration status of patients with HBV CHB 3 AHB patients and 13 patients after antiviral therapy on 14 integration sites. The integration sites of reads supports more than 2 of the conventional PCR and Sanger sequencing to verify, and detects the presence of the 14 integration sites in all 18 hepatitis B patients.
Results: high throughput sequencing results showed that the positive rate of integration of the 18 HBV patients with HBV was 100%, the total number of sites for 2083 patients with integration, the average integration site was 138.2 + 379.9 / person, including primary failure patients with HBV mean the largest number of integration sites (248.5 + 57.3 / person), to achieve sustained VR in the (18.6 + 13.7 / person). Patients with hepatitis B HBV integration site number and level of liver cell cccDNA, and the average coverage of liver cell HBsAg significantly positive correlation integral (P0.0001) to verify the success rate of the 14 integration sites is 100%.Sanger sequencing, 18 hepatitis B patients were Chr16:51320015. integration site
Conclusion: the positive rate of HBV integration is 100% in hepatitis B patients. The number of integration sites and the average coverage of patients are positively correlated with the HBsAg score of hepatocytes. The integration site Chr16:51320015 is the major integration site (MIS).
Fourth part: quantitative detection of HBV integrated virus host gene chimerism transcriptional expression and integration site Chr16:51320015
Objective: To study the transcriptional expression of the virus host gene of 14 HBV integration sites and to detect the integration site Chr16:51320015.
Methods: reverse transcription PCR was used to detect the transcriptional expression of virus host gene chimeras of 14 HBV integration sites. The copy number Chr16:51320015 of 18 cases of hepatitis B was detected by molecular cloning and real-time PCR.
Results: the study showed that 1,3,4,5,6,12,14 transcription and expression of chimeric chimeras, integration sites of transcription and expression of virus host genes, which in addition to 4 HBV S for the integration of the Regional District, No. 6 C District, the rest are X.18 Chr16:51320015 integration sites mean patients for 1.46 * 10-2 + 4.94 * 10-2copies/cell, which the maximum value is 0.212copies/cell, the minimum value is 3.48x1O-5copies/cell.
Conclusion: the transcription of virus host gene is closely related to the integration of HBx gene. HBsAg can be duplicated by integration of S region to produce.18, and Chr16:51320015 integration sites are all highly copy amplified.

【學(xué)位授予單位】：武漢大學(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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本文編號(hào)：1515047