Objective: To investigate the effect of high mobility group protein 1(HMGB1) on the proliferation and cytokine expression of human bone marrow mesenchymal stem cells (MSC).
Methods: Different concentrations of recombinant human HMGB1 protein (100, 200, 400, 800 and 1000 ng/ml) were incubated with MSC for 24, 48, 72 h and the proliferation of MSC were detected respectively by using the CCK-8 method and flow cytometry. The best concentrations of HMGB1 incubated with MSC was determined (200 ng/ml, 1000 ng/ml), and the flow cytomerty was used to determine the effect of HMGB1 on the proliferation of MSC. The mRNA expression levels of IL-10, TGF- β1, TSG-6 and IFN-γ in MSC incubated with HMGB1 protein were detected by real-time quantitative PCR and ELISA.
Results: The result of MSC identification and flow cytometry showed that the CD105, CD73 and CD90 were expressed, but did not expression CD45, CD34, CD11b, CD19 and HLA-DR; CCK-8 showed that HMGB1 at the concentrations of 100 ng/ml, 200 ng/ml and 400 ng/ml could promote the proliferation of MSC incubated for 24, 48 and 72 h as compared with the control group (P<0.05), and the most effective concentration was 200 ng/ml; flow cytometry showed that the compared with the control group, HMGB1 200 ng/ml could induce MSC from G1 phase to S phase to promote the proliferation of MSC; QPCR showed that the mRNA expression of MSC cytokines IL-10, TGF-β1, TSG-6 increased while IFN-γ decreased at the concentration of 200 ng/ml HMGB1 as compared with the control group. ELISA experiments showed that the HMGB1 200 ng/ml acting on MSC for 48 h could significantly promoted the secretion of IL-10, TGF-β 1 and TSG-6(P<0．05), while IFN-γ showed no significant difference as compared with control group.
Conclusion: Recombinant human HMGB1 can promote the proliferation and secretion of MSC in healthy people.
题目: HMGB1对健康人骨髓间充质干细胞增殖和细胞因子分泌的影响.
目的: 探讨高迁移率族蛋白1（HMGB1）对健康人骨髓间充质干细胞（MSC）增殖及相关细胞因子分泌的影响.
方法: 分别用CCK-8法和流式细胞术检测HMGB1对MSC增殖的影响，以不同浓度重组人HMGB1（100、200、400、800和1 000 ng/ml）分别作用MSC 24、48、72 h后，CCK-8法检测不同浓度的HMGB1对MSC增殖的影响。确定HMGB1对MSC作用的最佳实验药物浓度（200和1 000 ng/ml），进一步用流式细胞术明确HMGB1对MSC增殖的影响。采用ELISA与qPCR法检测HMGB1作用前后MSC中IL-10、TGF-β1、TSG-6、IFN-γ的表达水平.
结果: MSC的鉴定，流式细胞术检测结果显示，培养细胞表面表达CD105、CD73、CD90，不表达CD45、CD34、CD11b、CD19和HLA-DR。CCK-8法检测HMGB1对MSC增殖的影响发现，与对照组比较，HMGB1 100、200和400 ng/mL作用于MSC 24、48和72 h可明显促进MSC增殖（P<0.05），以200 ng/ml浓度作用的增殖效果最明显。流式细胞术检测结果显示，HMGB1于200 ng/ml时可诱导MSC由G1期进入S期，促进MSC增殖。qPCR法检测结果显示，在HMGB1 200 ng/ml作用下，MSC中IL-10、TGF-β1、TSG-6 mRNA较作用前表达增高，IFN-γ表达降低。ELISA实验表明，与对照组比较，HMGB1 200 ng/ml 作用48 h后，MSC分泌的细胞因子IL-10、TGF-β1、TSG-6蛋白水平显著升高（P<0.05），IFN-γ蛋白水平无明显变化（P<0.05）.
结论: 重组人HMGB1可促进健康人MSC增殖和影响其细胞因子分泌.