高迁移率族蛋白B1上调PC12细胞α7型乙酰胆碱受体表达的研究

The effect of high mobility group box-I protein on expression of a 7 nicotinic acetylcholine receptor in PC12 cells

目的观察高迁移率族蛋白B1（highmobilitygroupbox-1protein，HMGB1）对PCI2神经细胞重要神经递质受体a7型乙酰胆碱受体（a7nicotinicacetylcholinereceptor，et7nAChR）表达的影响，明确HMGBI潜在的神经免疫调节功能。方法体外培养PCI2细胞，HMGBl刺激PCI2细胞后，采用Westernblot和RT—PCR检测HMGB1对PCI2细胞ctTnAChR蛋白水平和mRNA表达影响的时效-量效关系，并进一步采用流式细胞术进行验证。结果①与对照组相比，20ng／mL、100ng／mL、500ng／mLHMGBl组仅7nAChR蛋白表达水平显著上调（P〈0．01），且3个时间点结果一致，其中48h时间点500ng／mLHMGBl组ct7nAChR表达上调尤为明显；②与对照组相比，24h时间点100ng／mL、500ng／mLHMGBl组ct7nAChRmRNA表达明显上调（P〈0．01），48h时间点500ngmL剂量组其表达亦显著上调（P〈0．01），72h时间点20ng／mL、100ng／mL、500ng／mL剂量组a7nAChRmRNA表达水平均明显增强（P〈0．01）。③流式细胞结果进一步证实了HMGBl可明显上调PCI2细胞a7nAChR的表达（P〈0．01）。结论HMGBl以时间依赖和剂量依赖方式上调PCI2细胞ct7nAChR的表达，提示HMGBl可显著影响神经细胞重要功能受体的表达，OL7nAChR参与了中枢对炎性细胞因子的识别和反应过程。

Objective To investigate the effect of high mobility group box-1 protein （HMGB1） on expression of a7 nicotinic acetylcholine receptor （a7nAChR）. Methods PC12 cells were stimulated with HMGB1 for consecutive time at different concentrations, and then detected mRNA and protein expression of a7nAChR by RT-PCR, Western blot, respectively. Using flow cytometry, the influence of HMGB1 on the expression of a7nAChR was verified. Results HMGB1 obviously influenced the expression of a7nAChR in PC12 cells. Compared with control group, levels of a7nAChR protein expression were significantly increased following HMGB1 treatment at 20 ng/mt, 100 ng/ml and 500 ng/ml for 24 h, 48 h and 72 h （P〈0.01）, respectively. Compared with the control group, ct7nAChR mRNA expression was significantly up-regulated following HMGB1 treatment at 500 ng/ml dose for 24 h and 48 h （P〈0.01）. Moreover, a7nAChR mRNA was significantly up-regulated after HMGB1 treatment either at above concentrations for 24 h （P〈0.01）. Flow cytometry result further confirmed the up-regulation of expression of a7TnAChR in PC12 cells after stimulation with HMGB1 （P〈0.01）. Conclusion HMGB1 is able to up-regulate the expression of a7nAChR in PC12 cells in a time and dose-dependent manner. These data demonstrate that HMGB1 markedly affects the expression of functional receptor in neural cells, and a7 nAChR may be involved in the recognition and response to inflammatory cytokines in central nervous system.