骨髓间充质干细胞对大鼠肺泡巨噬细胞Toll样受体mRNA和蛋白表达的影响

目的 观察骨髓间充质干细胞(mesenchymal stem cells，MSCs)对肺泡巨噬细胞Toll 样受体(toll-like receptor，TLR2/4)基因和蛋白的影响，为探讨骨髓MSCs移植对早期炎症介质的影响及炎症级联反应的调控机制做铺垫。 方法 巨噬细胞分为PBS对照组、骨髓MSCs对照组、脂多糖(lipopolysaccharide，LPS)对照组和LPS+骨髓MSCs处理组，各组均取1，3，6，12 h时相点。流式细胞仪检测骨髓MSCs表面标记阳性细胞率和巨噬细胞TLR2/4蛋白的表达；RT-PCR方法检测各组不同时相点巨噬细胞TLR2/4 mRNA表达变化；ELISA法检测各组不同时相点上清液中TNF-α浓度变化。 结果 与PBS对照组、骨髓MSCs对照组比较，LPS对照组巨噬细胞TLR2/4mRNA和蛋白表达明显升高(P＜0.01)，1h时开始增高，12h时达到峰值；同时TNF-α浓度也明显增高(P＜0.01)，6h时TNF-α浓度达到峰值。与LPS对照组比较，LPS+ MSCs组巨噬细胞TLR2/4 mRNA和蛋白表达降低(P＜0.01)，TNF-α浓度也降低(P＜0.01)。 结论 LPS刺激后巨噬细胞TLR2/4 mRNA和蛋白表达升高，TNF-α浓度也升高，后者峰值时间出现略早，说明TLR2/4表达与TNF-α之间存在一个正反馈环；骨髓MSCs可以明显降低LPS刺激后巨噬细胞TLR2/4 mRNA和蛋白表达及TNF-α浓度，说明骨髓MSCs打破了上述正反馈环。

Effects of bone marrow-derived mesenchymal stem cells on mRNA and protein expression of toll like receptor of rat alveolus macrophages

Objective To investigate the effect of bone marrow-derived mesenchymal stem cells (MSCs) on gene and protein expression of toll-like receptor, TLR2/4 of the rat alveolus macrophages so as to discuss the effect of MSCs transplantation on the early inflammatory mediators and the regulatory mechanism of the inflammatory cascade.Methods The alveolus macrophages were randomly divided into the PBS control group (n = 24), MSCs control group (n = 24), lipopolysaccharide (LPS) control group (n=24) and LPS + MSCs treatment group (n =24).All groups were taken time-points of 1, 3,6, 12 hours.Positive cell rate of MSCs with surface mark and the expression level of TLR2/4 protein on macrophage were detected by flow cytometry.The mRNA level of TLR2/4 expression on macrophage was detected by RT-PCR and the concentrations of TNF-o in cell culture supernatants by ELISA at the different time points. Results Compared with the PBS control group and MSCs control group, TLR2/4 mRNA and protein levels were significantly up-regulated, markedly increased at one hour and peaked at 12 hours (P ＜0.01) and the TNF-α concentrations in cell culture supernatants were significantly up-regulated and peaked at 6 hours in the LPS control group (P ＜ 0.01).Compared with the LPS control group, mRNA and protein level of TLR2/4 and TNF-o concentrations in the cell culture supernatants of the LPS + MSCs group were markedly down-regulated (P ＜ 0.01).Conclusions With LPS stimulation, TLR2/4 mRNA and protein expressions as well as TNF-αt concentration increase, when the peak of the TNF-αt concentration appears slightly earlier, indicating that there may be a positive feedback loop between TLR2/4 expression and TNF-ot.MSCs can significantly reduce TLR2/4 mRNA and protein expressions as well as TNF-α concentration after LPS stimulation, indicating that MSCs can break up the positive feedback loop.