异基因大鼠骨髓间充质干细胞对脂多糖刺激活化后小鼠巨噬细胞分泌因子的影响

Effect of bone marrow mesenchymal stem cells from allogeneic rats on secretory factors released by mouse macrophages after activation with lipopolysaccharide

背景：间充质干细胞可支持造血，抑制或延缓异基因骨髓移植后移植物抗宿主病的发生，延长异体移植器官的存活时间，具有免疫调节功能，然而其具体的免疫调节机制仍不清楚。目的：探讨SD大鼠骨髓间充质干细胞对脂多糖刺傲活化后的BALB／c小鼠腹腔巨噬细胞分泌因子的影响。设计、时间及地点；细胞对照观察，体外实验，于2006-12／2007-10在解放军兰州军区总医院血液病研究所完成。材料：清洁级20～22周龄雌性SD大鼠1只用于制备骨髓间充质干细胞，18～20周龄雄性BALB／c小鼠4只用于制备腹腔巨噬细胞，脂多糖为Sigma公司产品。方法：大鼠骨髓间充质干细胞以1×10^5/孔种植于24孔板，贴壁后作为支持层，再以相同密度加入用巯基乙酸钠腹腔注射刺激法收集的小鼠腹腔巨噬细胞，用终浓度1mg／L的脂多糖刺檄活化18h，收集培养上清待测，此为小鼠腹腔巨噬细胞＋脂多糖＋大鼠骨髓间充质干细胞组的干预措施。同时设立小鼠腹腔巨噬细胞组、小鼠腹腔巨噬细胞＋脂多糖组作为对照。主要观察指标：培养上清中肿瘤坏死因子n、转化生长因子β、一氧化氮的含量。结果：与单纯巨噬细胞比较，加入脂多糖刺激后培养上清中肿瘤坏死因子α及一氧化氮含量明显升高（F=10．368，P〈0．05）；而当骨髓间充质干细胞存在时，培养上清中肿瘤坏死因子α和一氧化氮含量均明显减少（F=0．017，P〈0．05）。各组培养上清中转化生长因子β含量基本相似（P〉0．05）。结论：实验首次发现SD大鼠骨髓间充质干细胞可抑制脂多糖刺激后BALB／c小鼠腹腔巨噬细胞的活化。

BACKGROUND： Mesenchymal stem cells can be used for hematopoiesis, inhibiting or delaying the occurrence of graft versus host disease after allogenic bone marrow transplantation, for prolonging the survival time of allografts. Mesenchymal stem cells possess the function of immunoregulation, but its regulatory mechanism is not clear. OBJECTIVE： To explore the influence of bone marrow mesenchymal stem cells （BMSCs） from Sprague Dawley rats on BALB/c mouse macrophage secretory factors after lipopolysaccharide stimulation. DESIGN, TIME AND SETTING： The in vitro cell control experiment was performed at the Institute of Hematonosis, Lanzhou General Hospital of Lanzhou Military Area Command of Chinese PLA from December 2006 to October 2007. MATERIALS： One Sprague Dawley rat aged 20-22 weeks old was used to harvest BMSCs. Four BALB/c mice aged 18-20 weeks old were employed to collect peritoneal macrophages. Lipopolysaccharide was purchased from Sigma, USA, METHODS： Rat BMSCs were incubated in a 24-well plate at a density of 1×10^5 per well. After adherence, peritoneal macrophages collected by infusing sodium thioglycollate were added in the medium. 1 mg/L lipopolysaccharide was applied to activate the mixture for 18 hours. Supernatant was collected for determination. This was the intervention measure in the mouse peritoneal macrophage ＋ lipopolysaccharide ＋ rat BMSCs group. Simultaneously, we set mouse peritoneal macrophage group, mouse peritoneal macrophage ＋ lipopolysaccharide group served as controls. MAIN OUTCOME MEASURES： Tumor necrosis factor a, transforming growth factor 13 and nitric oxide contents in supernatants. RESULTS： Compared with the simple macrophages, tumor necrosis factor a and nitric oxide contents significantly increased after treatment of lipopolysaccharide （F =10.368, P 〈 0.05）, When BMSCs were added, tumor necrosis factor a and nitric oxide contents significantly decreased in supernatants （F=0.017, P 〈 0,05）. Transforming growth factor 13 contents were similar in each group （P 〉 0,05）, CONCLUSION： The first clinical study shows that in vitro Sprague Dawley rat BMSCs can inhabit the activation of BALB/c mouse peritoneal macrophages after stimulating with lipopolysaccharide,