| Objective To investigate the effect of bone marrow mesenchymal stem cells(BM MSCs) on HBV replication, with the participation of lymphocytes.Methods(1) 80-100 g BN rats were used in this study. The whole bone marrow culture/sidewall separation filter method were combined to isolate BM MSCs. The third passage of BM MSCs were incubated with fluorescence-labelled antibodies and the surface markers were detected by flow cytometry analyses. The differentiation abilities of BM MSCs into lipocytes and osteoblasts were detected as well.(2) Cell co- culture: Hep G 2.2.15 cell line in good condition was selected. BM MSCs and T lymphocyte cells were isolated from the bone marrow and spleen of BN rats, respectively, and co-cultured by Transwell Chambers. Five groups were designed for experiments, group1: splenic lymphocytes; group2: Hep G 2.2.15; group3: BM MSCs/ Hep G 2.2.15; group4: splenic lymphocytes/ Hep G 2.2.15; group5: splenic lymphocytes / BM MSCs /Hep G 2.2.15.(3) The cellular activities of lymphocytes and Hep G 2.2.15 cells were detected by using MTT method at 24 h, 48 h and 72 h. The levels of HBV DNA and HBV ccc DNA were detected by real-time polymerase chain reaction(PCR). T cell subset was measured by using fluorescence labeled antibodies and flow cytometry analyses. ELISA was used to detect the level of cytokines in the supernatant of cultured cells.Result(1) Rat BM MSCs were isolated successfully and cultured in vitro. Cells have the typical morphological character of BM MSCs. According to the cell surface markers analyses, CD29, CD90 and RT1 A were expressed at BM MSCs surface,with positive rates of 97.0%, 96.3% and 96.3%, respectively. CD34, CD45 and RTl B were not expressed and the negative rates were >95%. Under special culture medium,BM MSCs were able to differentiate into lipocytes(red lipid droplet within cytoplasm by Oil Red O staining) and osteoblasts(black calcium salt deposit by von Kossa staining).(2) Compared with the group of BM MSCs / Hep G 2.2.15 and splenic lymphocytes / Hep G 2.2.15, the levels of HBV DNA and HBV ccc DNA of splenic lymphocytes / BM MSCs / Hep G2.2.15 were significant decreased at 48 h, HBV DNA:(181.000 ± 14.731)IU/m L vs(6270.000 ± 300.450) IU/m L,(2564.000 ±231.058)IU/m L,(2433.300 ± 302.379)IU/m L; HBV ccc DNA:(4.330×105 ± 0.464×105)IU/m L vs(11.100×105 ± 0.375×105) IU/m L,(8.930×105 ± 0.778×105) IU/m L,(9.850×105 ± 0.810×105) IU/m L,P<0.01.(3) The ratio of CD4+/CD8+ was increased and the expression of CD8+ cells was also down-regulated. Furthermore the level of IFN-? secretion was negatively correlated with HBV DNA levels, while the level of of IL-10 secretion was positively correlated with HBV DNA levels.Conclusion BM MSCs were isolated and cultured in vitro successfully. The cells were validated to be BM MSCs by cellular morphology, cell surface markers and differentiating potential in vitro. BM MSCs can inhibit the expression of HBV DNA, improve their ability to remove HBV by balancing type Tc1/Tc2 cells and their autocrine, and regulate the level of cytokine expression. |
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