Zheng Wei-Ping, Zhang Bo-Ya, Shen Zhong-Yang, Yin Ming-Li, Cao Yi, Song Hong-Li
Department of Organ Transplantation, Tianjin First Central Hospital, Tianjin 300192, P.R. China.
Tianjin First Central Hospital Clinic Institute, Tianjin Medical University, Tianjin 300070, P.R. China.
Mol Med Rep. 2017 May;15(5):2551-2559. doi: 10.3892/mmr.2017.6330. Epub 2017 Mar 15.
The aim of the present study was to explore the effects of co‑culturing bone marrow‑derived mesenchymal stem cells (BM-MSCs) cultured with hepatitis B virus (HBV)‑infected lymphocytes in vitro. BM‑MSCs and lymphocytes from Brown Norway rats were obtained from the bone marrow and spleen, respectively. Rats were divided into the following five experimental groups: Group 1, splenic lymphocytes (SLCs); group 2, HepG2.2.15 cells; group 3, BM‑MSCs + HepG2.2.15 cells; group 4, SLCs + HepG2.2.15 cells; and group 5, SLCs + BM‑MSCs + HepG2.2.15 cells. The viability of lymphocytes and HepG2.2.15 cells was assessed using the MTT assay at 24, 48 and 72 h, respectively. Levels of supernatant HBV DNA and intracellular HBV covalently closed circular DNA (cccDNA) were measured using quantitative polymerase chain reaction. Supernatant cytokine levels were measured by enzyme‑linked immunosorbent assay (ELISA). T cell subsets were quantified by flow cytometry using fluorescence‑labeled antibodies. In addition, the HBV genome sequence was analyzed by direct gene sequencing. Levels of HBV DNA and cccDNA in group 5 were lower when compared with those in group 3 or group 4, with a significant difference observed at 48 h. The secretion of interferon‑γ was negatively correlated with the level of HBV DNA, whereas secretion of interleukin (IL)‑10 and IL‑22 were positively correlated with the level of HBV DNA. Flow cytometry demonstrated that the percentage of CD3+CD8+ T cells was positively correlated with the levels of HBV DNA, and the CD3+CD4+/CD3+CD8+ ratio was negatively correlated with the level of HBV DNA. Almost no mutations in the HBV DNA sequence were detected in HepG2.2.15 cells co‑cultured with BM‑MSCs, SLCs, or in the two types of cells combined. BM‑MSCs inhibited the expression of HBV DNA and enhanced the clearance of HBV, which may have been mediated by the regulation of the Tc1/Tc2 cell balance and the mode of cytokine secretion to modulate cytokine expression.
本研究的目的是探讨体外共培养骨髓间充质干细胞（BM-MSCs）与乙型肝炎病毒（HBV）感染的淋巴细胞的效果。分别从骨髓和脾脏获取棕色挪威大鼠的BM-MSCs和淋巴细胞。大鼠被分为以下五个实验组：第1组，脾淋巴细胞（SLCs）；第2组，HepG2.2.15细胞；第3组，BM-MSCs + HepG2.2.15细胞；第4组，SLCs + HepG2.2.15细胞；第5组，SLCs + BM-MSCs + HepG2.2.15细胞。分别在24、48和72小时使用MTT法评估淋巴细胞和HepG2.2.15细胞的活力。使用定量聚合酶链反应测量上清液HBV DNA和细胞内HBV共价闭合环状DNA（cccDNA）的水平。通过酶联免疫吸附测定（ELISA）测量上清液细胞因子水平。使用荧光标记抗体通过流式细胞术对T细胞亚群进行定量。此外，通过直接基因测序分析HBV基因组序列。与第3组或第4组相比，第5组中HBV DNA和cccDNA的水平较低，在48小时时观察到显著差异。干扰素-γ的分泌与HBV DNA水平呈负相关，而白细胞介素（IL）-10和IL-22的分泌与HBV DNA水平呈正相关。流式细胞术表明，CD3+CD8+ T细胞的百分比与HBV DNA水平呈正相关，而CD3+CD4+/CD3+CD8+比值与HBV DNA水平呈负相关。在与BM-MSCs、SLCs共培养的HepG2.2.15细胞或两种细胞组合中，几乎未检测到HBV DNA序列的突变。BM-MSCs抑制HBV DNA的表达并增强HBV的清除，这可能是通过调节Tc1/Tc2细胞平衡和细胞因子分泌模式来调节细胞因子表达介导的。
