骨髓间充质干细胞在淋巴细胞参与下对 HBV 作用的体外研究

In vitro effects of bone marrow mesenchymal stem cells on HBV replication with the participation of lymphocytes

目的：探讨骨髓间充质干细胞（ bone marrow mesenchymal stem cells，BM MSCs）在体外淋巴细胞参与下对乙型肝炎病毒（ hepatitis B virus，HBV）复制的影响及其机制。方法选择培养状态良好的HepG2．2．15细胞，提取BN大鼠BM MSCs及新鲜脾淋巴细胞做共培养。实验分5组，分别于培养24 h、48 h及72 h后，采用MTT检测脾淋巴细胞及HepG2．2．15细胞活性，实时定量PCR测定HBV DNA及HBV cccDNA水平，流式细胞术检测T细胞亚群比例变化， ELISA检测细胞因子水平。结果在体外细胞培养的环境下，HBV DNA及HBV cccDNA在24 h、48 h及72 h时，与单纯HepG 2．2．15细胞组、BM MSCs＋HepG2．2．15细胞组及脾淋巴细胞＋HepG2．2．15细胞组比较，脾淋巴细胞＋BM MSCs＋HepG2．2．15细胞组表达水平明显降低，且在48 h 时差异有统计学意义， HBV DNA：（181．000±177;14．731） IU／ml vs （6270．000±177;300．450） IU／ml、（2564．000±177;231．058） IU／ml、（2433．300±177;302．379） IU／ml；HBV cccDNA：（4．330±215;105±177;0．464±215;105） IU／ml vs （11．100±215;105±177;0．375±215;105） IU／ml、（8．930±215;105±177;0．778±215;105） IU／ml、（9．850±215;105±177;0．810±215;105） IU／ml（P＜0．01）。细胞因子：IFN-γ与HBV DNA水平呈负相关，IL-10及IL-22分泌水平与HBV DNA水平呈正相关。 T细胞亚群：CD4＋／CD8＋升高，CD8＋细胞百分率与HBV DNA水平呈正相关。结论 BM MSCs可以抑制HBV DNA的表达，提高对HBV的清除能力，是通过调节Tc1／Tc2平衡和自身分泌方式，进而调节细胞因子表达水平实现的。

Objective To investigate the in vitro effects of bone marrow mesenchymal stem cells （BM MSCs） on the replication of HBV with the participation of lymphocytes and to analyze the possible mechanism.Methods The HBV genomic DNA transfected HepG2.2.15 cell line was used to evaluate the HBV replication.Bone marrow and spleen samples were collected from BN rats for the isolation of BM MSCs and T lymphocyte cells, respectively.Five groups of co-culturing with different cells were designed in this study.The cellular activities of lymphocytes and HepG2.2.15 cells were detected at the time of 24 h, 48 h and 72 h after co-culturing by using MTT method.The levels of HBV DNA and HBV cccDNA were detected by real-time polymerase chain reaction （ PCR） .T cell subsets in co-culture were measured by using fluores-cence labeled antibodies and flow cytometry analysis.ELISA was used to detect the levels of cytokines in the supernatant of cultured cells.Results Compared with HepG2.2.15 cells group, BM MSCs＋HepG2.2.15 cells and splenic lymphocytes＋HepG2.2.15 cells co-culture groups, the levels of HBV DNA and HBV cccDNA were significantly decreased in splenic lymphocytes＋BM MSCs＋HepG2.2.15 cells co-culture group after 48 h of culture [ HBV DNA： （ 181.000 ±177;14.731 ） IU/ml vs （ 6270.000 ±177;300.450 ） IU/ml,＆amp;nbsp;（2564.000±177;231.058） IU/ml, （2433.300±177;302.379） IU/ml;HBV cccDNA： （4.330±215;105 ±177;0.464±215;105 ） IU/ml vs （11.100±215;105±177;0.375±215;105） IU/ml, （8.930±215;105±177;0.778±215;105） IU/ml, （9.850±215;105±177;0.810±215; 105） IU/ml;P＆lt;0.01].The secretion of IFN-γin the supernatant of co-cultured cells was negatively corre-lated with HBV DNA level, but the levels of IL-10 and IL-22 were positively correlated with HBV DNA.The ratio of CD4＋/CD8＋cells was increased in splenic lymphocytes＋BM MSCs＋HepG2.2.15 cells co-culture group.The percentage of CD8＋cells showed a positive correlation with HBV DNA.Conclusion BM MSCs could inhibit the expression of HBV DNA to enhance the clearance of HBV strains.It might be possibly due to rebalancing of Tc1/Tc2 cells and regulating the expression of autocrine agents and cytokines.