大肠杆菌脂多糖对小鼠骨髓间充质干细胞炎症因子分泌的影响

Effects of Escherichia coli lipopolysaccharide on the secretion of inflammatory factors in mouse bone marrow mesenchymal stem cells

目的:探讨大肠杆菌脂多糖(lipopolysaccharide,LPS)对小鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)炎症细胞因子分泌的影响。方法:全骨髓贴壁法提取并培养小鼠BMSCs,CCK-8法检测LPS对BMSCs的毒性作用,用LPS(1μg/mL)激发小鼠BMSCs炎症反应,继续培养1 h,8 h,24 h后,qPCR法检测细胞白细胞介素1β(interleukin-1β,IL-1β),白细胞介素10(interleukin-10,IL-10),肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)mRNA的表达水平;Western Blot法分析细胞内IL-1β,IL-10,TNF-α蛋白的表达情况;ELISA法分析细胞上清中IL-1β,IL-10,TNF-α蛋白的表达情况。结果:CCK-8结果显示1μg/mL LPS处理对BMSCs无毒性作用;qPCR结果显示TNF-α,IL-1β,IL-10的表达均有不同程度增加,其中TNF-αmRNA的表达在诱导1 h后便显著上调,随后逐渐下降,IL-10和IL-1βmRNA的表达在1~8 h内逐渐上调,随后(8~24 h)下降;Western Blot结果显示:TNF-α和IL-1β蛋白的表达在1~8 h内增加,随后减少,IL-10蛋白的表达在24 h内逐渐增加;ELISA结果显示:TNF-α蛋白的表达在1~8 h内均显著增加,随后逐渐减少,IL-1β,IL-10蛋白的表达在24 h内均逐渐增加。结论:LPS刺激上调了BMSCs促炎因子IL-1β,TNF-α,抑炎因子IL-10的表达,不同细胞因子的表达在时间上存在先后顺序,这可能与BMSCs通过自身免疫调节机制(促炎/抑炎轴)分泌细胞因子来调节炎症过程有关。

Objective:To investigate the effects of E.coli lipopolysaccharide(LPS)on the secretion of inflammatory cytokines in mouse bone marrow mesenchymal stem cells(BMSCs).Methods:The primary BMSCs were cultured from bone marrow tissue from the tibia and femur of mice.CCK-8 assay was used to determine the toxic effect of LPS on BMSCs.LPS(1μg/mL)was used to induce the inflammatory response of BMSCs.After culturing for 1 h,8 h,24 h,fluorescence quantitative PCR assay was used to detect cellular interleukin-1β(IL-1β),interleukin-10(IL-10),tumor necrosis factor-α(TNF-α)mRNA.Western Blot assay was used to analyze the expression of IL-1β,IL-10,TNF-αprotein in cells.ELISA assay was used to detect the secretion of IL-1β,IL-10,TNF-αprotein in the cell supernatant.Results:CCK-8 results showed that 1μg/mL LPS had no toxic effect on BMSCs.The results of fluorescence quantitative PCR showed that the expression of TNF-α,IL-1β,and IL-10 mRNA all increased to varying degrees.Among them,the expression of TNF-αmRNA was significantly up-regulated after 1 hour of induction,and then gradually decreased.The expression of IL-10 and IL-1βmRNA gradually increased within 1~8 h,and then decreased slowly(8~24 h).Western-Blot results showed,the expression of TNF-αand IL-1βprotein increased within 1~8 h and then decreased.The expression of IL-10 protein gradually increased within 24 h.ELISA results showed that the expression of TNF-αprotein increased significantly within 1~8 h,and then gradually decreased.The expression of IL-1βand IL-10 protein both increased in the first 24 h.Conclusion:LPS stimulation up-regulates the expression of pro-inflammatory factors IL-1β,TNF-α,and anti-inflammatory factor IL-10 in BMSCs.The expression of different cytokines has a sequential order in time,which may be related to the regulation of inflammation by BMSCs secreting cytokines through autoimmune regulatory mechanisms(pro-inflammatory/anti-inflammatory axis).