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目的:观察甲基苯丙胺(methamphetamine,Meth)对原代小胶质细胞的损伤及炎性相关基因表达的影响。方法:原代培养SD胎鼠小胶质细胞,利用MTT和原位末端转移酶标记技术(TUNEL)分别检测Meth引起小胶质细胞活力和凋亡的变化。采用实时荧光定量聚合酶链反应法(real-time PCR)观察Meth对小胶质细胞炎性相关因子mRNA表达的影响。ELISA及试剂盒法检测Meth作用后小胶质细胞白介素(interleukin,IL)-6、肿瘤坏死因子(tumor necrosis factor,TNF)-α、诱导型一氧化氮合酶(inducible nitric oxide synthase,i NOS)的分泌改变。结果:MTT实验显示,Meth降低小胶质细胞活力,呈浓度依赖性,浓度为200μmol/L时与对照组比较,差异有统计学意义(P<0.01)。TUNEL实验结果显示200μmol/L Meth可引起细胞凋亡,与对照组比较,差异有统计学意义(P<0.05)。q-PCR结果显示Meth作用于小胶质细胞24 h可降低IL-24、一氧化氮合酶3(nitric oxide synthase 3,NOS3)表达水平,上调Peli3、Sigma受体1(Sigma receptor 1,Sig1-R)、IL-1β、IL-6、Toll样受体4(Toll like receptor 4,TLR4)的表达水平,差异有统计学意义(P<0.05)。此外,蛋白水平亦发现,Meth可促进IL-6和TNF-α的分泌。结论:Meth可降低小胶质细胞活力,诱导小胶质细胞凋亡,并引起IL-1β、IL-1R、IL-6、TLR4等炎性因子mRNA表达变化,促进IL-6和TNF-α的分泌,进而可能损伤中枢神经系统,产生神经毒性。
Objective: To observe the effect of methamphetamine (Meth) on the injury of primary microglia and the expression of inflammatory related genes. Methods: Primary cultured fetal rat microglia cells were cultured and the changes of microglial activity and apoptosis induced by Meth were detected by MTT assay and TUNEL assay. The effect of Meth on microglial inflammatory factor mRNA expression was observed by real-time PCR. The levels of interleukin (IL) -6, tumor necrosis factor (TNF) -α, inducible nitric oxide synthase (iNOS) ) Secretion changes. Results: MTT assay showed that Meth decreased the microglial activity in a concentration-dependent manner. Compared with the control group, Meth had a statistically significant difference (P <0.01) at a concentration of 200 μmol / L. TUNEL results showed that 200μmol / L Meth Meth could induce cell apoptosis, the difference was statistically significant (P <0.05) compared with the control group. The result of q-PCR showed that the effect of Meth on microglial cells for 24 h decreased the expression of IL-24, NOS3 and up-regulated the expression of Peli3, Sigma receptor 1 (Sig1 R, IL-1β, IL-6 and Toll-like receptor 4 (TLR4), the difference was statistically significant (P <0.05). In addition, the protein level also found that Meth can promote the secretion of IL-6 and TNF-α. Conclusion: Meth can reduce microglial viability, induce microglial apoptosis and induce changes of mRNA expression of IL-1β, IL-1R, IL-6 and TLR4 and promote the expression of IL-6 and TNF- Secretion, which may damage the central nervous system, resulting in neurotoxicity.