Detection of hepatitis viruses in dendritic cells by in situ PCR.

我们已经报道了乙型肝炎病毒（HBV）转基因小鼠（Tg）脾树突状细胞功能缺陷，这是一种人类慢性HBV携带者状态的小鼠模型。此外，树突状细胞功能障碍在人类丙型肝炎病毒感染中的作用已被证实。然而，树突状细胞功能缺陷的机制尚不清楚，也不清楚树突状细胞是否感染了肝炎病毒，还是树突状细胞作为这些病毒复制的载体。在此背景下，通过PCR和原位PCR杂交检测HBV感染患者树突状细胞和外周血单核细胞（PBMC）中HBV- dna的存在。为了优化实验条件，我们首先以HBV-Tg患者脾脏单个核细胞为阳性对照，进行了一系列初步实验。该方法可在HBV-Tg脾细胞中重复性检测HBV-DNA。HBV-DNA在几乎所有的脾细胞核中都可见。接下来，从慢性乙型肝炎患者身上获得树突状细胞和PBMC，经过8天的GM-CSF和IL-4富集程序，从外周血中分离出树突状细胞。从慢性乙型肝炎患者的树突状细胞和PBMC中提取DNA，并使用HBV特异性PCR引物进行PCR。在慢性肝炎患者的树突状细胞和PBMC中均检测到HBV-DNA。然而，这种方法在人树突状细胞和PBMC中不能稳定地检测到HBV-DNA，因为这些细胞的结构在原位PCR方法中被扭曲。需要进一步的研究来阐明HBV对人类HBV携带者树突状细胞的感染性。乙型肝炎病毒在树突状细胞中存在的影响还需要原位PCR和免疫学研究。然而，我们的数据提供了直接证据，证明慢性乙型肝炎患者的人外周血树突状细胞要么被HEV感染，要么HEV附着在树突状细胞上

We have already reported the defective function of splenic dendritic cells in hepatitis B virus (HBV)-transgenic mouse (Tg), a murine model of human chronic HBV-carrier state. Moreover, a role of dysfunction of dendritic cells have been shown in human hepatitis C virus infection. However, the mechanism underlying the functional defects of dendritic cells could not be clarified in full detail and it is unknown whether dendritic cells are infected with hepatitic viruses or dendritic cells act as a vehicle for the replication of these viruses.In this context, existence of HBV-DNA in dendritic cells and peripheral blood mononuclear cells (PBMC) was examined in patients infected with HBV by PCR and in situ PCR hybridization. In order to optimize the experimental conditions, first we used mononuclear cells from spleen of HBV-Tg as positive controls and undertook a series of preliminary experiments. HBV-DNA was reproducibly detected in HBV-Tg spleen cells using this methods. HBV-DNA was visua … More lized in almost all of the nuclei of spleen cells from HBV-Tg.Next, dendritic cells and PBMC were obtained from patients with chronic hepatitis B. Dendritic cells were isolated from the peripheral blood following a 8 days enrichment procedure in GM-CSF and IL-4. DNA was extracted from dendritic cells and PBMC from patients with chronic hepatitis B, and PCR was done using PCR primers specific for HBV. HBV-DNA was detected in both dendritic cells and PBMC from patients with chronic hepatitis. However, HBV-DNA was not stably detectable in human dendritic cells and PBMC by this method because the structure of these cells were distorted during the manipulation of cells for in situ PCR methodology.Further study is needed to clarify the infectivity of dendritic cells by HBV in human HBV-carriers. The effect of existence of HBV in dendritic cells also need in situ PCR and immunological investigations. However, our data has provided a direct evidence that human peripheral blood dendritic cells is either infected with HEV or HEV is attached to dendritic cells in patients with chronic hepatitis B. Less