【作者】 陈勇；

【导师】 秦波；

【作者基本信息】 重庆医科大学 ， 内科学， 2008， 硕士

【摘要】 目的:建立实时荧光定量PCR检测HBV cccDNA方法,测定重型乙型肝炎患者PBMC中HBV cccDNA含量,并进一步探索人工肝支持系统对重型乙型肝炎患者PBMC中HBV cccDNA的影响。方法:用不同浓度质粒标准品建立实时荧光定量PCR标准曲线,并检测该方法的灵敏性及重复性;HBV DNA阳性的慢性乙型肝炎（轻度）患者血清标本提取物经Plasmid_Safe^TMATP_dependent DNase酶切纯化后进行实时荧光定量PCR反应,验证该方法的特异性。以50例重型乙型肝炎患者人工肝血浆置换治疗前后PBMC作为检测标本,用已建立的实时荧光定量PCR方法对HBV cccDNA含量进行定量检测。用SAS8.1统计软件对结果进行统计学分析。结果:成功建立了HBV cccDNA实时荧光定量PCR检测方法,线性范围为1×10²～2.77×10⁹拷贝/ml。经特异性酶切消化后慢性乙型肝炎（轻度）患者血清中未检测到HBV cccDNA,重型乙型肝炎患者PBMC中存在HBV cccDNA。在人工肝血浆置换前后PBMC中HBV cccDNA含量（中位数log10值）分别为3.73,3.54（n=50,P>0.05）。血清总HBV DNA与PBMC中HBV cccDNA之间有较好的相关性（r=0.39,P<0.01）。总HBV DNA水平与PT（r=0.37,P<0.01）、ALT（r=0.29,P<0.05）、AST（r=0.39,P<0.01）水平呈正相关;HBV cccDNA水平与PT（r=0.34,P<0.05）、ALT（r=0.34,P<0.05）、AST（r=0.40,P<0.01）水平呈正相关。结论:本研究结果显示,所建立的实时荧光定量PCR方法具有良好的线性范围、灵敏度、特异性及重复性。重型乙型肝炎患者PBMC中存在HBV cccDNA。血清总HBV DNA与PBMC中HBV cccDNA呈正相关;总HBV DNA、HBV cccDNA与PT、ALT、AST呈正相关,说明总HBV DNA、HBV cccDNA均能在一定程度上反映肝脏损伤程度且有较好的一致性。在人工肝血浆置换后重型乙型肝炎患者PBMC中HBV cccDNA含量无明显降低。更多还原

【Abstract】 Objective:To establish a new fluorescence-based quantitative PCR assay for detecting hepatitis B virus covalently closed circular DNA（HBV cccDNA）.To explore the effect of artificial liver support system（ALSS）on HBV cccDNA in PBMC of patients with fulminant hepatitis B.Methods:Plasmids carrying HBV fragment were amplified with the specific primer set and fluorescent probe to establish the standard curve and to determine the sensitivity and reproducibility of the method.The extractive of HBV DNA positive sera samples from patients with chronic hepatitis B（mild）by were amplified simultaneously to test the specificity of the established fluorescence-based quantitative PCR method.Furthermore, the PBMC specimens from 50 patients of fulminant hepatitis B prior to and after treatment with artificial liver support system were collected.The genomic DNAs were extracted from PBMC of these patients and were further purified by Plasmid_Safe^TMATP_dependent DNase to remove HBV relaxed circular DNA（rc DNA）and the other form of HBV DNA except HBV cccDNA.Then HBV cccDNA was quantitatively detected by fluorescent PCR with specific primer set and Taqman probe.And the results were analyzed by SAS8.1 statistics software.Results : We successfully established a new fluorescence-based quantitative PCR method for HBV cccDNA with the linear range from 1×102 to 2.77×109copies per milliliter.HBV cccDNA was not observed in HBV DNA positive sera sample from patients with chronic hepatitis B （mild）which were purified by Plasmid_SafeTMATP_dependent DNase.HBV cccDNA was detectable in PBMC of the patients with fulminant hepatitis B. The level of HBV cccDNA in PBMC decreased not significantly following plasma exchange in artificial liver support system.The levels of total HBV DNA and HBV cccDNA had positive correlation with degree of PT、ALT、AST.Total HBV DNA also had positive correlation with HBV cccDNA.Conclusion:This study established a fluorescence-based quantitative PCR method for detecting HBV cccDNA, with wide linear range,high sensitivity,specificity and reproducibility.HBV cccDNA existed in PBMC of patients with fulminant hepatitis B. The level of total HBV DNA and HBV cccDNA has positive correlation with degree of PT、ALT and AST which might be indication of hepatocyte damage.We find that the level of HBV cccDNA in PBMC after plasma exchange in ALSS decrease without significantly.更多还原