Yan Qin, Lan Ying-Hua, Huang Yan-Xin, Fan Rong-Shan, Liu Lan, Song Shu-Peng, Li Yong-Guo
Department of Infectious Diseases, The First Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang 150001, P.R. China.
Mol Med Rep. 2016 Apr;13(4):3581-7. doi: 10.3892/mmr.2016.4973. Epub 2016 Mar 3.
Increasing evidence indicates that the hepatitis B virus (HBV) replicates in peripheral blood mononuclear cells (PBMCs), but at a low level. The present study aimed to establish a reliable and sensitive method that effectively detects HBV viral products for monitoring antiviral therapy, organ transplantation screening, and diagnosing occult HBV infection. In the present study, PBMCs (obtained from six healthy volunteers) were inoculated with HBV, and cultured with phytohemagglutinin (PHA) and interleukin‑2 (IL‑2) to stimulate cell proliferation. PBMCs were harvested, and quantitative detection of HBV DNA in cell suspension and intracellular hepatitis B surface antigen (HBsAg) was conducted on days 0, 1, 6 and 12, respectively. In situ hybridization, immunohistochemistry and reverse transcription‑polymerase chain reaction (RT‑PCR) were performed to analyze the HBV infection. The results demonstrated that HBV DNA increased concurrently with proliferation of PBMCs isolated from three of six healthy volunteers, and the mean number of PBMCs on day 12 was 13.61 times higher than the initially seeded cell number (P<0.01). The mean copies of HBV DNA at day 12 were 2.98 times higher compared with initial levels (P<0.05). Furthermore, intracellular HBsAg levels increased concurrently with proliferation of PBMCs in one group of cultured PBMCs, which was accompanied by increased HBV DNA levels. In addition, HBV nucleic acids were detected in PBMCs using in situ hybridization. Intracellular HBsAg was observed in PBMCs and HBV RNA was also detected by RT‑PCR. The present study demonstrated that HBV replicates in proliferating PBMCs, which were induced by PHA and IL‑2. This method offers a novel investigative tool to detect HBV infection in PBMCs and to monitor the course of HBV infection.
越来越多的证据表明，乙型肝炎病毒（HBV）在外周血单个核细胞（PBMC）中复制，但水平较低。本研究旨在建立一种可靠且灵敏的方法，以有效检测HBV病毒产物，用于监测抗病毒治疗、器官移植筛查以及诊断隐匿性HBV感染。在本研究中，将HBV接种于（从6名健康志愿者获取的）PBMC，并与植物血凝素（PHA）和白细胞介素-2（IL-2）共同培养以刺激细胞增殖。分别在第0、1、6和12天收获PBMC，并对细胞悬液中的HBV DNA和细胞内乙型肝炎表面抗原（HBsAg）进行定量检测。采用原位杂交、免疫组织化学和逆转录-聚合酶链反应（RT-PCR）分析HBV感染情况。结果显示，从6名健康志愿者中的3名分离出的PBMC增殖时，HBV DNA同时增加，且第12天PBMC的平均数量比最初接种的细胞数量高13.61倍（P<0.01）。第12天HBV DNA的平均拷贝数相较于初始水平增加了2.98倍（P<0.05）。此外，在一组培养的PBMC中，细胞内HBsAg水平随PBMC增殖而增加，同时伴有HBV DNA水平升高。另外，通过原位杂交在PBMC中检测到HBV核酸。在PBMC中观察到细胞内HBsAg，并且通过RT-PCR也检测到了HBV RNA。本研究表明，HBV在由PHA和IL-2诱导增殖的PBMC中复制。该方法为检测PBMC中的HBV感染以及监测HBV感染进程提供了一种新的研究工具。
