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

我们已经报道了在人类慢性乙肝病毒携带者状态的小鼠模型--乙肝病毒转基因小鼠(TG)中，脾树突状细胞的功能缺陷。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明。然而，树突状细胞功能缺陷的机制尚不完全清楚，目前尚不清楚树突状细胞是否感染了肝炎病毒，或者树突状细胞是复制这些病毒的载体。为了优化实验条件，我们首先以HBVTG的脾单个核细胞作为阳性对照，进行了一系列的初步实验。用该方法可重复性地检测到HBVTg脾细胞中的HBVDNA。乙肝病毒脱氧核糖核酸为…其次，从慢性乙肝患者的外周血中分离出树突状细胞和外周血单核细胞，经过8天的GM-CSF和IL-4的浓缩后，从外周血中分离出树突状细胞。从慢性乙型病毒性肝炎患者树突状细胞和外周血单核细胞中提取DNA，用针对乙肝病毒的特异性聚合酶链式反应进行聚合酶链式反应。慢性肝炎患者树突状细胞和外周血单核细胞中均检测到HBVDNA。但这种方法在人树突状细胞和PBMC中检测不到稳定的HBVDNA，这是因为原位聚合酶链式反应在操作细胞过程中这些细胞的结构发生了扭曲，还需要进一步的研究来阐明乙肝病毒携带者树突状细胞的感染性。树突状细胞中存在乙肝病毒的影响还需要原位聚合酶链式反应和免疫学研究。然而，我们的数据提供了一个直接证据，表明在慢性乙肝患者中，人类外周血树突状细胞要么感染了HEV，要么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