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目的:观察常压高浓度氧对脂多糖诱导N9小胶质细胞Toll受体4、TNF-α表达时序变化,初步探讨高氧对小胶质细胞促炎反应的作用及调控机制。方法:体外培养N9小胶质细胞,随机分为6组(n=3):空气组、sLPS组、hLPS组、高氧组、高氧+sLPS组、高氧+hLPS组。LPS浓度为100 ng/mL(sLPS)、1 mg/L(hLPS)。高氧(900 mL/L)暴露时间分别为2 h、6 h、12 h、16 h、24 h。RT-PCR检测TLR4的表达时序变化;Western blot检测处理12 h后各组TLR4蛋白表达;ELISA检测各组处理6 h、12 h、16 h、24 h培养上清中TNF-α的含量。结果:RT-PCR显示hLPS组在6 h后、sLPS组在16 h后TLR4 mRNA均表达上调,并随LPS浓度增加、暴露时间延长逐渐升高(P<0.05),24 h时hLPS组表达最高。6 h后在各个时间点高氧+hLPS组与hLPS比较TLR4 mRNA下调(P<0.05),在16 h、24 h最为明显(P<0.05)。Westernblot检测12 h高氧+sLPS组、高氧+hLPS组TLR4蛋白水平均低于相应浓度的LPS组(P<0.05)。ELISA结果示在各个时间点高氧+sLPS组、高氧+hLPS组与相应浓度的LPS组比较TNF-α均明显上调(P<0.05)。结论:高浓度氧暴露促进LPS诱导N9小胶质细胞的促炎症反应,TLR4可能参与此过程的负向调控。
OBJECTIVE: To observe the changes of Toll receptor 4 and TNF-α expression induced by lipopolysaccharide (LPS) in lipopolysaccharide (N9) microglia at normal pressure and high oxygen concentration, and to explore the effect of hyperbaric oxygen on the microglial pro-inflammatory response and its regulatory mechanism. Methods: N9 microglia cells were cultured in vitro and randomly divided into 6 groups (n = 3): air group, sLPS group, hLPS group, hyperoxia group, hyperoxia + sLPS group and hyperoxia + hLPS group. LPS concentrations of 100 ng / mL (sLPS), 1 mg / L (hLPS). Exposure time of high oxygen (900 mL / L) was 2 h, 6 h, 12 h, 16 h, 24 h respectively. The expression of TLR4 was detected by RT-PCR. The expression of TLR4 protein in each group was detected by Western blot 12 h later. The content of TNF-α in supernatant of 6 h, 12 h, 16 h and 24 h was measured by ELISA. Results: RT-PCR showed that TLR4 mRNA was up-regulated in sLPS group at 16 h after 6 h treatment, and prolonged with prolonged LPS concentration (P <0.05). The expression of TLR4 mRNA in hLPS group was highest at 24 h . After 6 h, the expression of TLR4 mRNA was down-regulated in the hyperoxia + hLPS group compared with the hLPS group at each time point (P <0.05), and was most obvious at 16 h and 24 h (P <0.05). The levels of TLR4 protein in the hyperoxia + hLPS group were lower than those in the corresponding LPS group (P <0.05) by Western blot in 12 h hyperoxia + sLPS group. The results of ELISA showed that TNF-αin hyperoxia + sLPS group and hyperoxia + hLPS group were significantly up-regulated compared with LPS group (P <0.05) at all time points. CONCLUSION: High concentration oxygen exposure can promote LPS-induced proinflammatory responses of N9 microglia cells. TLR4 may be involved in the negative regulation of this process.