HBV感染患者外周血单个核细胞HBV DNA和cccDNA的检测

目的 探讨乙型肝炎患者外周血单个核细胞（peripheral blood mononuclear cells，PBMC）能否感染乙型肝炎病毒（hepatitis B virus，HBV）并进行复制。方法 选取 137例住院患者作为研究对象，采集血液标本，分离提取外周血单个核细胞，使用细胞甩片法固定细胞，采用实时荧光定量聚合酶链式反应技术（real-time quantitative polymerase chain reaction，QPCR）和滚环扩增结合原位跨缺口原位聚合酶链式反应技术检测HBV共价闭合环状DNA（covalently closed circular DNA，cccDNA）；免疫组织化学染色法检测HBV DNA和HBV cccDNA表达。结果 137例标本中，68.6%（94/137）的标本经免疫组化染色后检测出HBV DNA的表达；83.9%（115/137）的标本原位杂交和滚环扩增PCR后检出HBV cccDNA阳性信号；血清HBV DNA浓度高于106 copies/ml的患者PBMC中HBV cccDNA阳性率为95.1%，低于106 copies/ml的阳性率为51.4%。结论 高浓度外周血HBV DNA是HBV感染外周血单个核细胞的危险因素，通过原位聚合酶链式反应方法能够更灵敏地检测HBV DNA和cccDNA在PBMC中的表达，为进一步研究证实PBMC感染导致HBV母婴传播途径的分子机制奠定基础。

Detection of ccc DNA and HBV DNA in peripheral blood mononuclear cells of patients with HBV infection by in situ hybridization

Objective To investigate whether hepatitis B virus (HBV) can infect and copy in the peripheral blood mononuclear cell (PBMC) in patients with chronic hepatitis B. Methods 137 inpatient attenders' peripheral blood was selected;mononuclear cells were isolated from peripheral blood;cells were fixed using cell rejection slice method; HBV DNA and cccDNA were detected with Real-time Quantitative polymerouse chain rection(QPCR) and rolling circle amplification combined with in situ cross gap PCR immunohistochemical staining method was used to detect HBV DNA and HBV cccDNA expression in PBMC. Results In 137 cases of specimen, after immunohistochemical staining, 68.6% (94/137) cases were detected the expression of HBV DNA, 83.9% (115/137) cases were deteced of HBV cccDNA positive signal by in situ hybridization and rolling circle amplification PCR; the positive rate of HBV cccDNA in PBMC of patients with serum HBV DNA concentration with higher than 106 copies/ml was 95.1%, the positive rate of less than 106 copies/ml was 51.4%. Conclusions HBV can infect the peripheral blood mononuclear cells and replicate in it, this study lays the foundation for further study of the PBMC infection leading to intrauterine infection of HBV.