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首页> 外文期刊>Hepatitis Monthly >Simple and Reliable Method to Quantify the Hepatitis B Viral Load and Replicative Capacity in Liver Tissue and Blood Leukocytes
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Simple and Reliable Method to Quantify the Hepatitis B Viral Load and Replicative Capacity in Liver Tissue and Blood Leukocytes

机译：简单可靠的方法来定量乙肝病毒在肝组织和血白细胞中的病毒载量和复制能力

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Background: A functional cure of chronic hepatitis B (CHB) is feasible, but a clear view of the intrahepatic viral dynamics in each patient is needed. Intrahepatic covalently closed circular DNA (cccDNA) is the stable form of the viral genome in infected cells, and represents the ideal marker of parenchymal colonization. Its relationships with easily accessible peripheral parameters need to be elucidated in order to avoid invasive procedures in patients. Objectives: The goal of this study was to design, set up, and validate a reliable and straightforward method for the quantification of the cccDNA and total DNA of the hepatitis B virus (HBV) in a variety of clinical samples. Patients and Methods: Clinical samples from a cohort of CHB patients, including liver biopsies in some, were collected for the analysis of intracellular HBV molecular markers using novel molecular assays. Results: A plasmid construct, including sequences from the HBV genome and from the human gene hTERT, was generated as an isomolar multi-standard for HBV quantitation and normalization to the cellular contents. The specificity of the real-time assay for the cccDNA was assessed using Dane particles isolated on a density gradient. A comparison of liver tissue from 6 untreated and 6 treated patients showed that the treatment deeply reduced the replicative capacity (total DNA/cccDNA), but had limited impact on the parenchymal colonization. The peripheral blood mononuclear cells (PBMCs) and granulocytes from the treated and untreated patients were also analyzed. Conclusions: A straightforward method for the quantification of intracellular HBV molecular parameters in clinical samples was developed and validated. The widespread use of such versatile assays could better define the prognosis of CHB, and allow a more rational approach to time-limited tailored treatment strategies.
机译：背景：功能性治愈慢性乙型肝炎（CHB）是可行的，但需要清楚了解每位患者的肝内病毒动力学。肝内共价闭合环状DNA（cccDNA）是病毒基因组在感染细胞中的稳定形式，并且代表实质定居的理想标记。需要阐明其与易于获取的外围参数的关系，以避免患者进行侵入性手术。目的：本研究的目的是设计，设置和验证可靠的，直接的方法，用于量化各种临床样品中乙型肝炎病毒（HBV）的cccDNA和总DNA。患者和方法：收集来自一组慢性乙型肝炎患者的临床样本，包括一些肝活检样本，以使用新型分子测定法分析细胞内HBV分子标记。结果：产生了一个质粒构建体，包括来自HBV基因组和人类基因hTERT的序列，作为等摩尔的多标准品，用于HBV定量和细胞内容物标准化。使用在密度梯度上分离的Dane颗粒评估cccDNA实时测定的特异性。比较了6名未接受治疗的患者和6名接受治疗的患者的肝组织，结果表明该疗法极大地降低了复制能力（总DNA / cccDNA），但对实质定植的影响有限。还分析了来自治疗和未治疗患者的外周血单核细胞（PBMC）和粒细胞。结论：建立并验证了一种用于定量临床样品中细胞内HBV分子参数的简单方法。此类多功能测定法的广泛使用可以更好地确定CHB的预后，并为时限定制的治疗策略提供更合理的方法。

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