炎症微环境对大鼠间充质干细胞增殖及Sirt6表达水平的影响

目的:分析沉默信息调控因子6(silent information regulator 6,Sirt6)在大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)中的表达情况,以及炎症微环境对其表达水平的影响,为研究Sirt6在牙周炎症条件下骨形成中的作用提供理论依据。方法:根据实验分为脂多糖(lipopolysaccharide,LPS)诱导组、对照组和LPS过表达Sirt6组。经10μg/mL LPS刺激6、12、24、48、72 h,建立细胞的炎症微环境;CCK-8法检测细胞的增殖水平;酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)检测炎性因子肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-1β(interleukin-1β,IL-1β)、白细胞介素-6(interleukin-6,IL-6)的表达;免疫荧光技术检测Sirt6的分布情况;通过反转录-聚合酶链反应(RT-PCR)技术、蛋白质印迹法(western blot)检测Sirt6在大鼠BMSCs中信使RNA(mRNA)及蛋白的表达水平。结果:10μg/mL LPS刺激BMSCs后,成功建立炎症微环境;与对照组相比较,CCK-8法检测到炎症微环境中BMSCs的增殖速率由增高逐渐趋于下降,炎症因子抑制了BMSCs的增殖速率(P<0.05),而过表达Sirt6则升高BMSCs的增殖速率(P<0.05);ELISA结果显示,随着时间的推移,炎症微环境中的TNF-α、IL-1β、IL-6含量逐渐下降(P<0.05),表明BMSCs对炎性因子具有调控作用;炎症微环境中,免疫荧光染色显示Sirt6的表达未受到明显抑制;RT-PCR结果显示,LPS过表达Sirt6组中,Sirt6的mRNA表达水平显著升高,且在不同时间点内Sirt6的mRNA表达水平呈现显著性升高;Western blot结果显示,同一时间点LPS诱导组中Sirt6蛋白的表达均出现下降,差异具有统计学意义(P<0.05),而且随着时间的推移,Sirt6的蛋白表达并没有出现显著性下降,差异无统计学意义(P>0.05)。结论:炎症因子可以通过抑制Sirt6蛋白的表达影响BMSCs的增殖作用,BMSCs中Sirt6也具有调控炎症因子的作用。

Effects of Inflammatory Microenvironment on Proliferation and Sirt6 Expression in Rat Mesenchymal Stem Cells

Objective:To address the expression of silent information regulator 6(Sirt6)in rat bone marrow mesenchymal stem cells(BMSCs)and the changes under inflammatory microenvironment.Methods:The experiment was divided into LPS induced group,control group and LPS over expressed Sirt6 group.BMSCs inflammatory microenvironment was established by stimulation with 10μg/mL lipopolysaccharide(LPS)for 6 h,12 h,24 h,48 h and 72 h.Cell proliferation level was detected with CCK-8.Expression of the inflammatory factors tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β)and interleukin-6(IL-6)was detected by enzyme linked immunosorbent assay(ELISA).Distribution pattern of Sirt6 was detected by immunofluorescence assay.Expression of Sirt6 mRNA and protein in rat BMSCs was detected by RT-PCR and western blot respectively.Results:Inflammatory microenvironment was established successfully by stimulating BMSCs with 10μg/mL LPS.CCK-8 detected changes in the proliferation rate of BMSCs from increasing in the control group to gradually decreasing in the inflammatory microenvironment(P<0.05).While Sirt6 overexpression increased the proliferation rate of BMSCs(P<0.05).ELISA detected that TNF-α,IL-1βand IL-6 in the inflammatory microenvironment gradually decreased over time(P<0.05).In immunofluorescence assay,expression intensity of Sirt6 in the inflammatory microenvironment was not significantly inhibited.RT-PCR detected significantly increased Sirt6 mRNA expression in LPS over expressed Sirt 6 group,with significantly increased Sirt6 mRNA expression at each time point.Western blot showed that in LPS induced group,Sirt6 protein expression was decreased at each time point compared to the control group,with statistically significant differences(P<0.05),and did not decrease significantly over time,without statistically significant differences(P>0.05).Conclusion:Inflammatory factors could affect the proliferation rate of BMSCs by inhibiting expression of Sirt6 protein.Sirt6 also has regulatory effect on the inflammatory factors in BMSCs.