乙肝患者肝内cccDNA水平、HBV整合状况及其临床意义研究
发布时间：2018-02-16 08:05
本文关键词： HBV整合 共价闭合环状DNA 高通量测序 适时定量聚合酶链反应 出处：《武汉大学》2014年博士论文 论文类型：学位论文
【摘要】：乙型肝炎病毒(HBV)感染是严重威胁人民生命健康的疾病。乙肝感染肝炎迁移不愈、并可能发展为肝细胞癌(HCC)的根源在于肝细胞核内cccDNA池的持续存在及HBV整合入宿主细胞。我们采用改进后的灵敏、特异、简便的SYBR Green I RT-PCI(?)法及高通量序列捕获测序技术分别对急性自限性乙肝患者(AHB)、抗病毒治疗不同预后慢性乙肝患者(CHB)的肝细胞cccDNA水平及HBV整合情况进行检测,确定可代表CHB治愈的肝细胞cccDNA阈值,了解乙肝患者HBV整合的一般情况。 第一部分：cccDNA检测方法学的建立 目的：建立灵敏、特异、简便的肝细胞HBV cccDNA检测方法。 方法：从PSAD酶切、荧光采集温度、肝组织DNA提取等方面对传统cccDNA SYBR Green I RT-PCR检测法进行改进。 结果：改进后的cccDNASYBR Green I RT-PCR检测方法简便,特异性好,灵敏度高,其检测下限值达24copies/μl。 结论;简便、灵敏、特异的肝细胞cccDNA检测方法的建立为临床大量乙肝患者肝细胞cccDNA检测奠定了基础。 第二部分：肝细胞内cccDNA水平对抗病毒治疗CHB患者预后的预测价值研究 目的：探讨肝细胞内cccDNA水平对抗病毒治疗CHB患者预后的预测价值及与患者病毒学、血清学、病理指标的相关性。 方法：对60名乙肝患者,包括11名AHB患者、46名接受抗病毒治疗CHB患者[21名原发性治疗失败、11名实现持续病毒学反应(VR)、15名实现持续VR和HBsAg血清学清除]及3名自身免疫性肝炎患者的肝细胞cccDNA和总DNA(tDNA)水平、血清HBV DNA、HBsAg、HBeAg和ALT水平进行检测。 结果：AHB患者肝细胞cccDNA和tDNA水平(分别为0.002copies/cell和0.04copies/cell)显著低于CHB患者。实现持续性VR和HBsAg血清学清除CHB患者的肝细胞cccDNA水平(0.012copies/cell)显著低于原发性治疗失败患者(4.18copies/cell, P㩳0.0001),但和实现持续性VR患者无显著差异(0.039copies/cell,阐.169)。实现持续性VR和HBsAg血清学清除CHB患者的肝细胞平均tDNA水平(0.096copies/cell)显著低于原发性治疗失败患者(371copies/cell, P㩳0.0001)及实现持续性VR患者(1.62copies/cell, P=0.001)。 ROC曲线分析显示肝细胞cccDNA水平(0.015copies/cell)和tDNA水平(0.23copies/cell)在预测实现持续性VR和HBsAg血清学清除方面没有显著差异(ROC曲线下面积分别为0.88和0.96,P0.10)。肝细胞cccDNA水平和血清ALT、HBeAg及肝细胞tDNA水平显著正相关(P=0.024,P=0.001和P0.0001),和血清HBV DNA无相关性(P=0.12),和血清HBeAg阴性及及HBeAg阳性患者血清HBsAg水平无相关性(P=0.84和P-0.146)。 结论：AHB和抗病毒治疗后实现持续性VR和HBsAg血清学清除的CHB患者肝细胞内存有极微量的cccDNA。肝细胞cccDNA水平检测有希望成为预测抗病毒治疗预后的可靠指标。 第三部分：高通量测序及Sanger测序检测乙肝患者HBV整合研究 目的：探讨乙肝携带者、AHB及抗病毒治疗CHB患者的HBV整合情况。 方法：采用高通量序列捕获测序技术检测2名乙肝携带者、3名AHB患者及13名抗病毒治疗后CHB患者HBV整合状况。对整合位点reads支持大于2个的14个整合位点用常规PCR和Sanger测序进行验证,并检测这14个整合位点在全部18名乙肝患者中的存在情况。 结果：高通量测序结果显示18名乙肝患者中HBV整合阳性率为100%,患者整合位点总数为2083个,平均整合位点为138.2±379.9个/人,其中原发性失败患者HBV平均整合位点数目最多(248.5±57.3个/人),实现持续性VR患者次之(18.6±13.7个/人)。乙肝患者HBV整合位点数目与肝细胞cccDNA水平、平均覆盖度及肝细胞HBsAg积分显著正相关(均为P0.0001)。Sanger测序法验证14个整合位点成功率为100%,18名乙肝患者均出现整合位点Chr16：51320015。 结论：乙肝患者HBV整合阳性率为100%,其整合位点数目和患者检测平均覆盖度、肝细胞HBsAg积分显著正相关。整合位点Chr16：51320015为广泛发生的主要整合位点(MIS)。 第四部分：HBV整合病毒-宿主基因嵌合体转录表达和整合位点Chr16：51320015定量检测研究 目的：研究14个HBV整合位点的病毒-宿主基因嵌合体转录表达,并对整合位点Chr16：51320015进行定量检测。 方法：采用反转录PCR法检测14个HBV整合位点的病毒-宿主基因嵌合体转录表达,采用分子克隆及荧光定量PCR法定量检测18例乙肝患者的整合位点Chr16:51320015拷贝数。 结果：嵌合体转录表达研究显示1、3、4、5、6、12、14号整合位点出现病毒-宿主基因嵌合体转录表达,其中除4号HBV整合区域为S区外、6号为前C区外,其余均为X区。18例患者整合位点Chr16：51320015均值为1.46×10-2±4.94×10-2copies/cell,其中最大值为0.212copies/cell,最小值为3.48x1O-5copies/cell。 结论：病毒-宿主基因转录表达与HBx基因整合密切相关,HBsAg可以由整合S区复制产生。18例Chr16：51320015整合位点均为高拷贝克隆扩增。
[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.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R512.62
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