摘要
目的:
Alzheimer's病(AD)是影响记忆和认知功能的进行性退化性神经病变,是痴呆最常见的原因。研究表明,β淀粉样蛋白(Aβ)增加是AD中枢病因性过程,凝聚态Aβ具有明确的神经毒性作用。但亦有研究提出,Aβ在纤维性沉积前即可溶性Aβ也可产生神经毒性作用。海马是学习和记忆的关键部位,直接参与记忆的获取和建立。海马CA3区与空间辨别性学习记忆关系密切。而学习和记忆存储的分子和生理机制研究表明,钾通道、蛋白激酶C、记忆相关蛋白Cp20、细胞内钙调节在学习和记忆机制中起重要作用,其中钾离子通道在学习和记忆中起着关键作用,提示钾通道在老年痴呆发病过程中具有重要意义。有研究认为可溶性Aβ_(25-35)对新生大鼠海马CA3区锥体神经元延迟整流钾电流(I_k)有明显抑制作用,其效应具有时间依赖性和电压依赖性,但具体作用机制不清。本实验以大鼠海马CA3区锥体神经元为研究对象,采用膜片钳与单细胞RT-PCR技术相结合的方法,研究可溶性Aβ_(25-35)对新生大鼠海马CA3区锥体神经元几种代表性延迟整流钾通道亚型mRNA表达的影响,从分子生物学的角度探讨I_k变化的分子机制。为研究Aβ在AD发病过程中的作用机制以及临床应用钾通道调节剂预防和治疗AD提供理论依据。
方法:
采用酶解和机械分离相结合的方法,急性分离新生大鼠海马CA3区锥体神经元。运用膜片钳,借助其电极形成的吸收通道提取单个细胞,然后对其进行单细胞水平的分子生物学研究。由于单个细胞中提取的mRNA数量很少,应用传统的单对引物对单细胞mRNA进行RT-PCR扩增,得到的产物结果不够理想,故本实验采用的巢式设计的两对PCR引物,进行两轮PCR扩增。RT-PCR扩增后凝胶电泳成像分析,观察可溶性Aβ_(25-35)对大鼠海马CA3区锥体神经元延迟整流钾离子通道亚型Kv1.2、Kv1.5、Kv2.1表达的影响。
结果:
1.延迟整流钾通道亚型Kv1.2,Kv1.5,Kv2.1在正常大鼠海马CA3区锥体神经元上均有不同程度的表达。其中,Kv2.1的表达水平较高。
2.加入浓度为1.0、2.5、5.0μM可溶性Aβ_(25-35)1分钟后,大鼠海马CA3区锥体神经元延迟整流钾通道亚型Kv1.2mRNA的相对表达量与对照组相比明显降低,分别降低15.9%(P＜0.01)、24.0%(P＜0.01)、35.8%(P＜0.01)。
3.加入浓度为1.0、2.5、5.0μM可溶性Aβ_(25-35)1分钟后,大鼠海马CA3区锥体神经元延迟整流钾通道亚型Kv1.5mRNA的相对表达量与对照组相比明显降低,分别降低17.8%(P＜0.01)、23.2%(P＜0.01)、36.1%(P＜0.01)。
4.加入浓度为1.0、2.5、5.0μM可溶性Aβ_(25-35)1分钟后,大鼠海马CA3区锥体神经元延迟整流钾通道亚型Kv2.1 mRNA的相对表达量与对照组相比明显降低,分别降低17.2%(P＜0.01)、23.7%(P＜0.01)、36.9%(P＜0.011。
5.加入浓度为5.0μM可溶性Aβ_(25-35)孵育1、3、5、8、10分钟后,大鼠海马CA3区锥体神经元延迟整流钾通道亚型Kv2.1 mRNA的相对表达量与对照组相比明显降低,分别降低了38.7%(P＜0.01)、43.5%(P＜0.01)、53.3%(P＜0.01)、65.6%(P＜0.01)、73.7%(P＜0.01)。
结论:
1.在正常新生大鼠海马CA3区锥体神经元细胞膜上,延迟整流钾通道亚型Kv1.2,Kv1.5,Kv2.1 mRNA均有不同程度的表达,其中以Kv2.1的表达较为丰富。
2.可溶性Aβ_(25-35)对大鼠海马CA3区锥体神经元延迟整流钾通道亚型Kv1.2,Kv1.5,Kv2.1 mRNA的表达有较明显的抑制作用,这种抑制作用具有浓度依赖性和时间依赖性。
3.可溶性Aβ_(25-35)对大鼠海马CA3区锥体神经元延迟整流钾通道亚型mRNA表达的抑制导致钾通道数量减少,这可能是其对延迟整流钾电流抑制作用的机制之一。
Object:
Alzheimer's disease(AD)is a neurodegenerative disorder influencing functions of memory and cognition,and AD is the most common reason of dementia.Some researches indicate that the main pathological characteristic of AD is increased amyloidβ-peptide(Aβ),and the deposition of Aβin the brain has clear neurotoxicity. There are other researches showing that soluble Aβalso has neurotoxicity. Hippocampus is a key structure of learning and memory,which is directly related to the acquisition and foundation of learning and memory.Hippocampal CA3 area correlates to learning memory of spatial discrimination closely.While researches on the molecule and physiology mechanism of learning and memory show that potassium channel、PKC、memory-relating protein Cp20 and regulation of intracellular Ca~(2+)play an important role in the mechanism of learning and memory, especially that potassium channel plays a key role,and this suggests that potassium channel has significant effects on AD.Some studies indicate that soluble Aβ_(25-35)can inhibit I_k of the neurons of hippocampal CA3 area distinctly in newborn rats.The effect is time-dependent and voltage-dependent,but the specific mechanism remains unknown.We investigate the effect of soluble Aβ_(25-35)on the expression of mRNA of several K~+ channel of hippocampal CA3 area of newborn rats,using neurons of hippocampal CA3 area and taking the methods of patch clamp and single cell RT-PCR and research the mechanism of I_k change in the point of molecular biology. Our research provided theoretical proof to the mechanism of Aβin the AD disease and the precaution and cure of AD.
Methods:
Combining the methods of enzymic digestion and mechanical separation,we separated rat hippocampal CA3 pyramidal neurons from the slice of the brain tissue. After that,we use the patch clamp technique to get a single cell,and use the RT-PCR technique to amplify potassium channels' mRNA.Then we can record the effects of soluble Aβ_(25-35)on the mRNA expression of delayed rectifier potassium channel subtypes Kv1.2,Kv1.5 and Kv2.1.
Results:
1.In the rat hippocampal CA3 pyramidal neurons,delayed rectifier potassium channel Kv1.2,Kv1.5 and Kv2.1 mRNA are expressed in different levels,and Kv2.1 has a relatively high level expression.
2.Compared to the control group,the expression level of kv1.2 mRNA fell 15.9% (P＜0.01)、24.0%(P＜0.01)and 35.8%(P＜0.01)after addition of 1.0、2.5 and 5.0μM soluble Aβ_(25-35)for 1 minute.
3.Compared to the control group,the expression level of kv1.5 mRNA fell 17.8% (P＜0.01)、23.2%(P＜0.01)and 36.1%(P＜0.01)after addition of 1.0、2.5 and 5.0μM soluble Aβ_(25-35)for 1 minute.
4.Compared to the control group,the expression level of kv2.1 mRNA fell 17.2% (P＜0.01)、23.7%(P＜0.01)and 36.9%(P＜0.01)after addition of 1.0、2.5 and 5.0μM soluble Aβ_(25-35)for 1 minute.
5.Compared to the control group,the expression level of kv2.1 mRNA fell 38.7% (P＜0.01)、43.5%(P＜0.01)、53.3%(P＜0.01)、65.6%(P＜0.01)、73.7%(P＜0.01) after addition of 5.0μM soluble Aβ_(25-35)for 1、3、5、8 and 10 minutes.
Conclusion:
1.In the rat hippocampal CA3 pyramidal neurons,delayed rectifier potassium channel Kv1.2,Kv1.5 and Kv2.1 mRNA are expressed in different levels,and Kv2.1 has a relatively high level expression.
2.The expression of Kv1.2、Kv1.5 and Kv2.1 mRNA were obviously inhibited by soluble Aβ_(25-35)and the inhibition effect was concentration- and time-depended.
3.The number of potassium channel reduced because of the block effect of soluble Aβ_(25-35)on rat hippocampal CA3 pyramidal neurons.And this may be one of the mechanisms how soluble Aβ_(25-35)can inhibit electric current of potassium.
引文
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