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目的:通过建立星形胶质细胞氨中毒的模型,探讨瞬时受体电位M2(TRPM2)阳离子通道在其中发挥的作用。方法:分离并培养小鼠原代星形胶质细胞。实验分为5组:对照组、氯化铵处理组、氯化铵+3-氨基苯甲酰胺(3-AB)处理组、氯化铵+PJ-34处理组和TRPM2基因敲除小鼠+氧化铵处理组。测定细胞的活性、caspase-3活性、细胞坏死和体积大小,以此来衡量氨中毒的程度。用全细胞膜片钳记录TRPM2通道电流变化。结果:氯化铵引起细胞肿胀伴随着细胞坏死。聚腺苷二磷酸核糖聚合酶(PARP)抑制剂3-AB和PJ-34抑制了氯化铵引起的阳离子电流,并减轻了氯化铵引起的相应细胞伤害。TRPM2基因敲除小鼠组细胞的伤害明显减轻(P<0.01)。结论:TRMP2通道的激活是细胞暴露于氯化铵后发生肿胀、坏死的必要步骤;氯化铵诱导的星形胶质细胞肿胀与坏死密切相关。
OBJECTIVE: To investigate the role of the transient receptor potential M2 (TRPM2) cation channel in establishing a model of astrocyte ammonia poisoning. Methods: Mouse primary astrocytes were isolated and cultured. The experiment was divided into five groups: control group, ammonium chloride treatment group, ammonium chloride + 3-aminobenzamide (3-AB) treatment group, ammonium chloride + PJ-34 treatment group and TRPM2 knockout mice + Ammonium oxide treatment group. Measure the cell activity, caspase-3 activity, cell necrosis and size, in order to measure the degree of ammonia poisoning. Whole cell patch clamp recording of TRPM2 channel current changes. Results: Ammonium chloride causes cell swelling with cell necrosis. Poly (ADP-ribose) polymerase (PARP) inhibitors 3-AB and PJ-34 inhibit the ammonium currents caused by ammonium chloride and reduce the corresponding cell damage caused by ammonium chloride. The injury of cells in TRPM2 knockout mice was significantly reduced (P <0.01). CONCLUSION: Activation of TRMP2 channel is a necessary step for cell swelling and necrosis after exposure to ammonium chloride. Ammonium chloride-induced astrocyte swelling is closely related to necrosis.