钾离子通道亚型Kv1.3参与甲基苯丙胺引起小胶质细胞的损伤

发布时间：2018-03-21 23:36

本文选题：甲基苯丙胺　切入点：小胶质细胞　出处：《南京医科大学》2013年硕士论文　论文类型：学位论文

【摘要】：目的观察甲基苯丙胺（Methamphetamine，Meth）对大鼠胎鼠小胶质细胞的损伤作用。方法原代培养SD胎鼠小胶质细胞，细胞计数试剂盒（CCK-8及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。结果MTT实验显示，甲基苯丙胺呈浓度依赖性（10、30、100、300、1000μM）降低小胶质细胞活力。在Meth浓度为10、30、100μM时，细胞活力有所下降，当浓度为300、1000μM时，Meth会造成细胞活力下降，差别有统计学差异（p0.05）。CCK-8实验，钾离子通道非特异性抑制剂Tetraethylamine（TEA）、4-Aminopyridine（4-AP）及钾离子通道亚型Kv1.3特异性抑制剂Margatoxin（MgTx）预孵后再加入Meth，Meth所引起的细胞损伤可被部分逆转。TUNEL实验可知，，100、300μM Meth可引起细胞凋亡，300μM时差异有统计学意义。结论Meth明显引起小胶质细胞损伤，该损伤过程可以部分被TEA、4-AP及MgTx等钾离子通道抑制剂逆转。目的观察甲基苯丙胺（Meth）引起小胶质细胞损伤中电压门控钾离子通道亚型Kv1.3、Kv1.5的作用。方法原代培养SD胎鼠小胶质细胞，采用蛋白质印迹法（western blot）和实时荧光定量聚合酶链反应法（RT-PCR）观察Kv1.3、Kv1.5表达变化情况。采用RT-PCR法观察Meth作用后小胶质细胞内炎性因子分泌情况。同时探讨Meth引起小胶质细胞内Kv1.3变化及Meth与p38-丝裂原活化蛋白激酶（MAPK）信号通路之间的关系。结果Meth引起Kv1.3mRNA和蛋白水平表达增高，Kv1.3特异性抑制剂MgTx可减少上述表达。Meth对Kv1.5mRNA和蛋白水平表达均无明显影响。Meth所致小胶质细胞内炎性因子表达量的增高亦可被MgTx所阻滞。Meth引起的小胶质细胞损伤可通过预孵MgTx降低，其损伤可以激活p38MAPK通路。结论Kv1.3可能参与甲基苯丙胺引起小胶质细胞损伤机，该通道抑制后，Meth引起的炎性因子表达显著降低。此外，Meth明显激活p38MAPK通路。因而，Kv1.3可能成为小胶质细胞损伤治疗的一个新靶点。
[Abstract]:Objective to observe the damage effect of methamphetamine on rat fetal microglia. Methods Primary culture of SD fetal rat microglia cells was performed. Cell count kit CCK-8 and MTT) and in situ terminal transferase labeling (Tunel) were used to detect the changes of microglia viability and apoptosis induced by Meth. Methamphetamine decreased the viability of microglia cells in a dose-dependent manner. When the concentration of Meth was 10 ~ 30100 渭 M, the cell viability was decreased, and when the concentration was 300 渭 M, the cell viability was decreased, and the difference was statistically significant (p 0.05N. CCK-8). Tetraethylamineine (Tetraethylamine), a nonspecific inhibitor of potassium channel, Tetraethylamine, 4-Aminopyridine 4-AP), and the potassium channel subtype Kv1.3 specific inhibitor, Kv1.3, were preincubated and then added with Methanemeth, which could be partially reversed. Tunel showed that 100300 渭 M Meth could induce apoptosis at 300 渭 M. Conclusion Meth can obviously induce microglia injury. This process can be partially reversed by potassium channel inhibitors such as tea 4-AP and MgTx. Objective to observe the effect of voltage-gated potassium channel subtype Kv1.3 Kv1.5 on microglia injury induced by methamphetamine method primary cultured SD fetal rat microglia cells. Western blot and RT PCR were used to observe the expression of Kv1.3 and Kv1.5. RT-PCR was used to observe the secretion of inflammatory factors in microglia after Meth treatment. Meanwhile, microglia were induced by Meth. The changes of Kv1.3 in cells and the relationship between Meth and p38 mitogen-activated protein kinase (p38 mitogen activated protein kinase) signal pathway. Results Meth induced the increase of Kv1.3mRNA and protein expression. MgTx, a specific inhibitor of Kv1.3, could reduce the expression of Kv1.5mRNA and protein. No significant effect was found on the expression of inflammatory cytokines in microglial cells induced by. Meth. Meth could also be blocked by MgTx. Meth-induced microglial injury could be reduced by preincubation of MgTx. Conclusion Kv1.3 may be involved in the mechanism of microglia injury induced by methamphetamine. The expression of inflammatory cytokines induced by Meth was significantly decreased after this channel was inhibited. In addition, Meth significantly activated the p38MAPK pathway. Therefore, Kv1.3 may be a new target for the treatment of microglia injury.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R114

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