慢性乙型肝炎患者外周血单个核细胞及肝组织中HBV cccDNA定量检测

Quantitative detection of hepatitis B virus cccDNA in Peripheral blood mononuclear cells and livers in patients with chronic hepatitis B

目的优化HBV共价闭合环状DNA（cccDNA）特异性定量检测方法，并观察HBV cccDNA在慢性乙型肝炎患者外周血单个核细胞（PBMC）及肝组织中的分布情况。方法标本抽提核酸后，以不降解质粒的ATP依赖的DNA酶（PSAD）进行酶切，再以跨双缺口的特异性引物进行荧光PCR定量检测HBV cccDNA；用10份HBV高滴度（10^7拷贝／mL）血清和梯度稀释的HBV克隆质粒标准品，验证此检测方法的特异度和灵敏度；取慢性乙型肝炎患者PBMC和肝组织穿刺标本各50份，检测其总HBVDNA和cccDNA。结果10份血清标本均无假阳性cccDNA检测结果，灵敏度可达到103拷贝／mL。所有PBMC中cccDNA检测均阴性；所有肝组织中总HBVDNA和cccDNA检测均阳性，总HBVDNA含量为3．19×10^3～6．69×10^7拷贝／μg人基因组DNA，cccDNA含量为7．32×10^0～6．51×10^6拷贝／μg人基因组DNA，同一患者肝组织中总HBVDNA含量是cccDNA含量的10．7倍至9．2×10^5倍。结论本方法特异度和灵敏度高。PBMC不能支持HBV的复制。在慢性乙型肝炎患者的肝组织中均可检测到cccDNA，但其水平并非与细胞内总HBV DNA水平正相关。

Objectives To optimize the specific quantitative assay for determining hepatitis B virus covalently closed circular DNA （ HBV cccDNA）, and to observe the existence and distribution of cccDNA in peripheral blood mononuclear cells（PBMC）and liver tissues in patients with chronic hepatitis B. Methods DNA was extracted from samples and then was treated with Plasmid-Safe ATP- dependent DNase（PSAD）to reduce the relaxed circular DNA（rcDNA）. The products were further amplified by real-time PCR with primers spanning the two direct repeat region of HBV genome. Ten sera samples with high level（10^7 copies/mL）HBV DNA and HBV plasmid standards with 1 ： 10 serial dilutions were used to investigate the specificity and sensitivity of this assay. The levels of HBV cccDNA in 50 PBMC samples and 50 liver biopsy samples from patients with chronic hepatitis were detected by the same assay. Results No significant fluorescent signal was observed when 10 sera samples with high level HBV DNA were amplified, and lowest limitation of this assay determined by 1 ： 10 serial dilutions of HBV cloned plasmid was 103 copies/mL. HBV cccDNA were undetectable in all PBMC samples, while both total HBV DNA and cccDNA were positive in all liver samples. The total HBV DNA levels in liver samples ranged from 3.19 ×10^2 to 6.69 × 10^7 copies/μg human genomic DNA, and the levels of cccDNA ranged from 7.32 × 10^0 to 6.51 × 10^6. The level of HBV DNA was 10.7 to 9.2 ×10^5 times higher than the level of cccDNA in the same liver sample. Conclusions This assay is highly specific and sensitive, which can be applied in the scientific research of HBV as well as in clinic examination. HBV cccDNA is not detected in PBMC, which do not support the assumpation that HBV could replicate in PBMC. In contrast, cccDNA is detected in all liver biopsy samples, which, although varying significantly in different patients, is not positively related to the level of in tracellular total HBV DNA.