第一部分：半胱氨酰白三烯受体拮抗剂孟鲁司特和HAMI3379在大鼠皮层神经元缺血性损伤中的抗氧化作用 第二部分：原代小胶
发布时间：2019-03-29 19:53
【摘要】：目的：在大鼠皮层神经元缺血性损伤模型中,探讨两种半胱氨酰白三烯受体(CysLT1R和CysLT2R)拮抗剂的抗氧化作用。 方法：以CysLT1R拮抗剂孟鲁司特和CysLT2R拮抗剂HAMI3379预处理,对体外培养大鼠皮层神经元进行缺糖缺氧／恢复(OGD/R)或H202处理,观察神经元活化氧(ROS)产生、线粒体膜电位以及神经元存活率变化,以及两种药物在这些过程中的作用。同时,以RNA干扰抑制CysLT1R和CysLT2R表达,观察以上变化。 结果：OGD处理1h后,神经元ROS产生增加,在恢复0.5h时达高峰；0.01-1μM孟鲁司特和HAMI3379能轻度但显著地抑制OGD/R诱导的ROS产生(P0.05)。OGD/R诱导神经元线粒体膜电位下降,0.01～1μM孟鲁司特有轻度抑制作用(P0.05),而HAMI3379无明显作用。外源性H202浓度和时间依赖性损伤神经元,孟鲁司特和HAMI3379在0.1～1μM浓度范围内,能轻度但显著地减轻H202诱导的损伤(P0.05)。CysLT1R siRNA和CysLT2R shRNA对以上反应均无显著影响。 结论：孟鲁司特和HAMI3379在神经元缺血性损伤中,均有轻度抗氧化作用,且可能是不依赖受体的作用。 目的：初步研究大鼠原代小胶质细胞半胱氨酰白三烯受体表达情况。 方法：取出生24h的SD新生鼠,从皮层组织分离、纯化原代小胶质细胞。通过免疫荧光染色方法,观察原代小胶质细胞半胱氨酰白三烯受体CysLT1、CysLT2受体以及GPR17的表达情况,确定是否存在共定位；并且,使用激动剂LPS、NMLTC4诱导,观察受体的表达变化。 结果：分离、纯化并获得原代小胶质细胞,免疫荧光染色结果显示,CysLT1、 CysLT2受体和GPR17都有表达,它们之间存在明显的共表达(即CysLT1R/CysLT2R、CysLT1R/GPR17和CysLT2R/GPR17)。LPS能刺激CysLT2受体发生内移,并有一定的聚集；而CysLT2受体激动剂NMLTC4诱导的CysLT2受体也诱导内移和聚集,但比LPS弱。 结论：原代小胶质细胞表达半胱氨酰白三烯受体CysLT1、CysLT2受体和GPR17,并存在明显的共定位；炎症诱导剂LPS能够诱导CysLT2受体聚集和内移,CysLT2受体选择性激动剂引起的CysLT2受体聚集和内移有待进一步确认。
[Abstract]:Aim: to investigate the antioxidation effects of two cysteinyl leukotriene receptor antagonists (CysLT1R and CysLT2R) in the rat model of cortical neuron ischemic injury. Methods: CysLT1R antagonist montelukast and CysLT2R antagonist HAMI3379 were pretreated with glucose deficiency hypoxia / recovery (OGD/R) or H2O2 to observe the production of activated oxygen (ROS) in cultured rat cortical neurons in vitro. Changes in mitochondrial membrane potential and neuronal viability and the role of both drugs in these processes. At the same time, the expression of CysLT1R and CysLT2R was inhibited by RNA interference, and the above changes were observed. Results: after treatment with OGD for 1 h, the production of ROS increased and reached the peak at 0.5 h. Montelukast and HAMI3379 at 0.01-1 渭 M inhibited ROS production induced by OGD/R (P0.05), decreased mitochondrial membrane potential induced by OGD / R and slightly inhibited montelukis specific to 0.01-1 渭 M (P0.05). HAMI3379 had no obvious effect. Exogenous H2O2 concentration and time-dependent damage to neurons, montelukast and HAMI3379 in the concentration range of 0.1 ~ 1 渭 M, slightly but significantly reduced the injury induced by H2O2 (P0.05). Both CysLT1R siRNA and CysLT2R shRNA had no significant effect on the above responses. Conclusion: both montelukast and HAMI3379 have mild antioxidant effect in neuronal ischemic injury, and may be receptor-independent. Aim: to study the expression of cysteinyl leukotriene receptor in primary microglia of rats. Methods: primary microglia were isolated from cortex of 24 h-old SD newborn mice. The expression of cysteinyl leukotriene receptor CysLT1,CysLT2 receptor and GPR17 in primary microglia was observed by immunofluorescence staining, and the co-localization was determined. In addition, the expression of cysteinyl leukotriene receptor was induced by LPS,NMLTC4. Results: primary microglia were isolated, purified and obtained. The results of immunofluorescence staining showed that both CysLT1, CysLT2 receptor and GPR17 were expressed, and there was obvious co-expression between them (that is, CysLT1R/CysLT2R,). CysLT1R/GPR17 and CysLT2R/GPR17). Lipopolysaccharide (LPS) can stimulate the inward migration and aggregation of CysLT2 receptors. CysLT2 receptors induced by NMLTC4, a CysLT2 receptor agonist, also induced inward migration and aggregation, but were weaker than those induced by LPS. Conclusion: the primary microglia express cysteinyl leukotriene receptor CysLT1,CysLT2 receptor and GPR17, and have obvious co-localization. LPS, an inflammatory inducer, can induce aggregation and inward migration of CysLT2 receptors. The aggregation and inward migration of CysLT2 receptors induced by selective agonists of CysLT2 receptors need to be further confirmed.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R965
本文编号：2449835
[Abstract]:Aim: to investigate the antioxidation effects of two cysteinyl leukotriene receptor antagonists (CysLT1R and CysLT2R) in the rat model of cortical neuron ischemic injury. Methods: CysLT1R antagonist montelukast and CysLT2R antagonist HAMI3379 were pretreated with glucose deficiency hypoxia / recovery (OGD/R) or H2O2 to observe the production of activated oxygen (ROS) in cultured rat cortical neurons in vitro. Changes in mitochondrial membrane potential and neuronal viability and the role of both drugs in these processes. At the same time, the expression of CysLT1R and CysLT2R was inhibited by RNA interference, and the above changes were observed. Results: after treatment with OGD for 1 h, the production of ROS increased and reached the peak at 0.5 h. Montelukast and HAMI3379 at 0.01-1 渭 M inhibited ROS production induced by OGD/R (P0.05), decreased mitochondrial membrane potential induced by OGD / R and slightly inhibited montelukis specific to 0.01-1 渭 M (P0.05). HAMI3379 had no obvious effect. Exogenous H2O2 concentration and time-dependent damage to neurons, montelukast and HAMI3379 in the concentration range of 0.1 ~ 1 渭 M, slightly but significantly reduced the injury induced by H2O2 (P0.05). Both CysLT1R siRNA and CysLT2R shRNA had no significant effect on the above responses. Conclusion: both montelukast and HAMI3379 have mild antioxidant effect in neuronal ischemic injury, and may be receptor-independent. Aim: to study the expression of cysteinyl leukotriene receptor in primary microglia of rats. Methods: primary microglia were isolated from cortex of 24 h-old SD newborn mice. The expression of cysteinyl leukotriene receptor CysLT1,CysLT2 receptor and GPR17 in primary microglia was observed by immunofluorescence staining, and the co-localization was determined. In addition, the expression of cysteinyl leukotriene receptor was induced by LPS,NMLTC4. Results: primary microglia were isolated, purified and obtained. The results of immunofluorescence staining showed that both CysLT1, CysLT2 receptor and GPR17 were expressed, and there was obvious co-expression between them (that is, CysLT1R/CysLT2R,). CysLT1R/GPR17 and CysLT2R/GPR17). Lipopolysaccharide (LPS) can stimulate the inward migration and aggregation of CysLT2 receptors. CysLT2 receptors induced by NMLTC4, a CysLT2 receptor agonist, also induced inward migration and aggregation, but were weaker than those induced by LPS. Conclusion: the primary microglia express cysteinyl leukotriene receptor CysLT1,CysLT2 receptor and GPR17, and have obvious co-localization. LPS, an inflammatory inducer, can induce aggregation and inward migration of CysLT2 receptors. The aggregation and inward migration of CysLT2 receptors induced by selective agonists of CysLT2 receptors need to be further confirmed.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R965
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