HIV相关性痴呆中gp120和甲基苯丙胺对外向钾电流的作用及其引起海马神经元损伤机制的研究
发布时间：2018-10-08 06:51
【摘要】：第一部分:TEA和4-AP敏感型钾通道参与由gp120引起的海马神经元的凋亡 虽然HIV病毒壳蛋白gp120对神经元的损伤作用在HIV相关性痴呆(HIV-associated demential,HAD)发展的过程中起到重要作用,但致其损伤的作用靶点仍未明确。因此,明确gp120引起中枢神经系统神经元损伤的作用靶点对于HAD的防治显得尤为重要。近期有报道称,钾通道可能是引起HAD的又一靶点,因为持续性的钾离子外流会引起细胞凋亡。因而,该实验中,通过运用全细胞膜片钳的实验方法,我们发现gp120能显著引起外向钾电流的增高,且呈明显的浓度依赖性。实验进一步表明,TEA和4-AP敏感型钾通道均参与gp120引起的钾电流增大,这一结果与MTT和TUNEL实验中,TEA和4-AP能有效阻断gp120引起海马神经元的毒性一致。以上结果表明:gp120引起TEA和4-AP敏感型钾电流的增大,促发钾离子的外流,最终导致神经元的凋亡。 第二部分:4-AP敏感型钾电流参与由甲基苯丙胺引起的海马神经元的凋亡 甲基苯丙胺(Methamphetamine,Meth)是目前广泛滥用的一种非法精神兴奋剂。Meth的滥用可引起神经元的损伤,例如皮层灰质的缺失,海马神经元的萎缩,但其机制仍不清楚。细胞体积与胞内钾离子的稳态密切相关,持续性的钾离子外流可引起细胞凋亡性体积缩小。该实验中,我们以原代培养胚胎大鼠的海马神经元作为研究对象,探索由Meth引起的神经元损伤与钾通道之间的关系。利用全细胞膜片钳的方法,我们发现,Meth能明显增加4-AP敏感型钾电流的电流密度,而对TEA敏感型钾通道作用不明显。与此结果一致的是,利用MTT和TUNEL的方法,我们发现4-AP能够部分阻断Meth引起的海马神经元的毒性作用。进一步研究表明,Meth增大4-AP敏感型钾电流可能是通过抑制ERK通路的磷酸化来实现的。 这些结果表明,Meth可能通过抑制ERK的磷酸化引起4-AP敏感型钾电流的增大,最终导致海马神经元的损伤。 第三部分甲基苯丙胺与gp120对海马神经元的综合作用 毒品的滥用是神经退行性疾病的重要危险因素,同时也是HIV感染的主要途径之一。毒品和HIV病毒的共同作用可能会造成更为严重的神经系统的损伤,加快HAD的发展。甲基苯丙胺(Meth)是目前最常见,使用最为广泛的毒品之一,研究表明,Meth对中枢神经系统有直接的毒性作用,可引起海马的皱缩,胶质细胞的激活等。基于第一部分gp120及第二部分Meth对海马神经元的损伤作用,我们推测,gp120可能会与Meth对神经元产生相加或协同的毒性作用,引起更为严重的细胞损伤。因此,我们利用全细胞膜片钳,MTT和TUNEL的实验方法,初步探索了gp120+Meth处理组与gp120或Meth单独处理组引起的海马神经元的损伤,但在该实验中,我们并未到观察到HIV病毒蛋白gp120与Meth对海马神经元的综合作用,其原因值得进一步探索。
[Abstract]:Part 1: tea and 4-AP sensitive potassium channels participate in the apoptosis of hippocampal neurons induced by gp120 although the damage of HIV virus capsid protein gp120 to neurons plays an important role in the development of HIV associated dementia (HIV-associated demential,HAD). However, the target of its injury is still unclear. Therefore, it is very important for the prevention and treatment of HAD to identify the target of neuronal injury induced by gp120. There have been recent reports that potassium channels may be another target for HAD, as persistent outflows of potassium lead to apoptosis. Therefore, by using whole-cell patch clamp method, we found that gp120 could significantly increase outward potassium currents in a dose-dependent manner. The results further showed that both tea and 4-AP sensitive potassium channels were involved in the increase of potassium currents induced by gp120, which was consistent with the effect of tea and 4-AP on blocking the toxicity of gp120 induced hippocampal neurons in MTT and TUNEL experiments. The above results indicate that the TEA and 4-AP sensitive potassium currents are increased and the efflux of potassium ions is stimulated by 1: gp120, which leads to the apoptosis of neurons. Part two: 4-AP sensitive potassium current involved in apoptosis of hippocampal neurons induced by methamphetamine (Methamphetamine,Meth) is a widespread abuse of illicit psychostimulant. Meth can cause neuronal damage. For example, the absence of gray matter in the cortex and atrophy of hippocampal neurons remain unclear. The volume of cells is closely related to the homeostasis of intracellular potassium ions, and the persistent outflow of potassium ions can reduce the apoptotic volume of cells. In this experiment, we investigated the relationship between neuronal damage induced by Meth and potassium channels in primary cultured hippocampal neurons of embryonic rats. By using whole-cell patch clamp method, we found that Meth could significantly increase the current density of 4-AP sensitive potassium current, but had no obvious effect on TEA sensitive potassium channel. Using MTT and TUNEL, we found that 4-AP partially blocked the toxicity of hippocampal neurons induced by Meth. Further studies indicate that the increase of 4-AP sensitive potassium currents may be achieved by inhibiting phosphorylation of ERK pathway. These results suggest that Meth may induce the increase of 4-AP sensitive potassium current by inhibiting the phosphorylation of ERK, and eventually lead to the injury of hippocampal neurons. The third part is the comprehensive effect of methamphetamine and gp120 on hippocampal neurons. Drug abuse is an important risk factor for neurodegenerative diseases and one of the main routes of HIV infection. The combined action of drug and HIV virus may cause more serious damage to nervous system and accelerate the development of HAD. Methamphetamine (Meth) is one of the most common and widely used drugs. Studies have shown that methamphetamine has a direct toxic effect on the central nervous system, which can cause hippocampal shrinkage and activation of glial cells. Based on the damage of gp120 and Meth to hippocampal neurons in the first part, we speculate that Gp120 and Meth may have additive or synergistic toxic effects on neurons, resulting in more serious cell damage. Therefore, using whole-cell patch clamp MTT and TUNEL methods, we preliminarily explored the hippocampal neuron damage induced by gp120 Meth treatment and gp120 or Meth alone, but in this experiment, We did not observe the comprehensive effect of HIV virus protein gp120 and Meth on hippocampal neurons.
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R363
本文编号：2255839
[Abstract]:Part 1: tea and 4-AP sensitive potassium channels participate in the apoptosis of hippocampal neurons induced by gp120 although the damage of HIV virus capsid protein gp120 to neurons plays an important role in the development of HIV associated dementia (HIV-associated demential,HAD). However, the target of its injury is still unclear. Therefore, it is very important for the prevention and treatment of HAD to identify the target of neuronal injury induced by gp120. There have been recent reports that potassium channels may be another target for HAD, as persistent outflows of potassium lead to apoptosis. Therefore, by using whole-cell patch clamp method, we found that gp120 could significantly increase outward potassium currents in a dose-dependent manner. The results further showed that both tea and 4-AP sensitive potassium channels were involved in the increase of potassium currents induced by gp120, which was consistent with the effect of tea and 4-AP on blocking the toxicity of gp120 induced hippocampal neurons in MTT and TUNEL experiments. The above results indicate that the TEA and 4-AP sensitive potassium currents are increased and the efflux of potassium ions is stimulated by 1: gp120, which leads to the apoptosis of neurons. Part two: 4-AP sensitive potassium current involved in apoptosis of hippocampal neurons induced by methamphetamine (Methamphetamine,Meth) is a widespread abuse of illicit psychostimulant. Meth can cause neuronal damage. For example, the absence of gray matter in the cortex and atrophy of hippocampal neurons remain unclear. The volume of cells is closely related to the homeostasis of intracellular potassium ions, and the persistent outflow of potassium ions can reduce the apoptotic volume of cells. In this experiment, we investigated the relationship between neuronal damage induced by Meth and potassium channels in primary cultured hippocampal neurons of embryonic rats. By using whole-cell patch clamp method, we found that Meth could significantly increase the current density of 4-AP sensitive potassium current, but had no obvious effect on TEA sensitive potassium channel. Using MTT and TUNEL, we found that 4-AP partially blocked the toxicity of hippocampal neurons induced by Meth. Further studies indicate that the increase of 4-AP sensitive potassium currents may be achieved by inhibiting phosphorylation of ERK pathway. These results suggest that Meth may induce the increase of 4-AP sensitive potassium current by inhibiting the phosphorylation of ERK, and eventually lead to the injury of hippocampal neurons. The third part is the comprehensive effect of methamphetamine and gp120 on hippocampal neurons. Drug abuse is an important risk factor for neurodegenerative diseases and one of the main routes of HIV infection. The combined action of drug and HIV virus may cause more serious damage to nervous system and accelerate the development of HAD. Methamphetamine (Meth) is one of the most common and widely used drugs. Studies have shown that methamphetamine has a direct toxic effect on the central nervous system, which can cause hippocampal shrinkage and activation of glial cells. Based on the damage of gp120 and Meth to hippocampal neurons in the first part, we speculate that Gp120 and Meth may have additive or synergistic toxic effects on neurons, resulting in more serious cell damage. Therefore, using whole-cell patch clamp MTT and TUNEL methods, we preliminarily explored the hippocampal neuron damage induced by gp120 Meth treatment and gp120 or Meth alone, but in this experiment, We did not observe the comprehensive effect of HIV virus protein gp120 and Meth on hippocampal neurons.
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R363
【参考文献】
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1 ;Roles of Chemokine Receptor 4 (CXCR4) and Chemokine Ligand 12 (CXCL12) in Metastasis of Hepatocellular Carcinoma Cells[J];Cellular & Molecular Immunology;2008年05期
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