【作者】 丁砚兵；

【导师】 凃晋文；

【作者基本信息】 湖北中医学院 ， 中医内科学， 2009， 博士

【摘要】 目的：运用膜片钳技术原理和全细胞记录方式研究寿尔智胶囊对健康大鼠海马神经元细胞及AD模型大鼠脑神经递质所表达的离子通道的影响，从而探讨中药防治老年性痴呆的机理。方法：（1）运用血清药理学方法，制备家兔含药血清。分为寿尔智含药血清组、哈伯因含药血清组和生理盐水血清组（简称空白组）。寿尔智含药血清组以川芎嗪（寿尔智组药物之一，川芎的有效成分）为代表成分进行定性检测。（2）运用神经元细胞分散及培养技术，分散和培养海马神经元细胞运用于膜片钳技术和全细胞记录方式记录离子通道电流。（3）运用离子通道的原理及膜片钳全细胞记录方式分别记录各组（加含药血清前后）海马神经元细胞钠离子、钾离子及L-型钙离子通道电流，并观察其Ⅰ-Ⅴ曲线的变化。（4）运用D-半乳糖眼球后注射方法复制AD模型大鼠，并运用分子克隆技术，神经递质基因表达系统及膜片钳技术记录AD模型大鼠脑mRNA在爪蟾卵母细胞上所表达的神经递质通道电流和跨膜电压，观察寿尔智含药血清对其跨膜电压和通道电流的影响。结果：（1）寿尔智含药血清中药代表成分川芎嗪定性检测结果表明兔血清中含有寿尔智方药中的药物成分。（2）分离后培养的海马神经元细胞生长良好，培养12-15天的海马神经元细胞贴壁完全，细胞体积较大，有利于高阻封接，在细胞记录中能观察到明显的突触自发活动。（3）寿尔智含药血清能有效增强钠离子通道电流峰值，加速钠离子通道失活；明显增强钾电流峰值，增加钾通道电导；有效抑制L-型钙离子电流峰值，与对照组比较有显著性差异（p＜0．05）。（4）寿尔智含药血清能增强AD模型大鼠脑mRNA在爪蟾卵母细胞上所表达的神经递质通道电流和跨膜电压，与对照组比较有显著性差异（p＜0．05）。结论：本实验研究表明寿尔智对于体外培养的大鼠海马神经元细胞钠离子、钾离子、L-型钙离子通道电流及其相关Ⅰ-Ⅴ曲线具有明显的影响。中药复方寿尔智能明显提高钠离子、钾离子电流I_Na、I_k的峰值，同时可使钠离子通道迅速失活，而增加钾离子通道电导，使海马神经元除极化时间缩短、迅速产生较强幅度的动作电位，抑制海马神经元细胞持续去极化，使其复极化时间缩短。寿尔智能有效抑制钙电流幅度，即具有明显的阻滞钙离子内流的作用，降低细胞内钙含量，防止因钙超载引起的细胞凋亡。研究寿尔智对AD模型大鼠脑神经递质所表达的离子通道电流的影响，其结果表明寿尔智能有效促进兴奋性神经递质释放。因此寿尔智通过上述作用机理产生了对海马神经元既兴奋神经细胞又节制其兴奋性神经递质过度释放的作用。其中主要以加速钠离子通道失活和抑制钙离子内流调节作用保护细胞不受兴奋性递质过度释放所产生的兴奋毒性作用的损伤，从而延长细胞的生命活力，因此，寿尔智具有明确的保护脑组织和增强记忆的作用，从而达到防治老年性痴呆（AD）的目的。更多还原

【Abstract】 Objective: the patch clamp technique in the whole cell recording was used to study the effect of Shou’ erzhi capsule on the hippocampus neurons cells of healthy rats and ion channel expressed by AD（Alzheimer disease） model rats’ cerebral neurotransmitters. Based on these experiments, the preventive and therapeutic mechanism of Alzheimer’s disease with traditional Chinese herbs has been studied.Methods: （1） prepared rabbits medicated serum by serum pharmacological method, and been divided into three groups, including Shou’ erzhi medicated serum, Haboine medicated serum and physiological saline medicated serum （blank group for short）. In Shou’ erzhi medicated serum group, ligustrazine （components of Shou’ erzhi capsule, and the effective composition of ligustra） was chosen to carry out qualitative detection as representative.（2） Dispersed and cultivated hippocampus neurons cells by utilizing neurons cells dispersion and culture techniques, which would be used to record ion channel current in the patch clamp technique and the whole cell recording.（3） Applying ion channel principle and the patch clamp technique in the whole cell recording to record the Na ion, K ion and L-Ca ion channel currents of hippocampus neurons cells （with medicated serum） respectively, and observing the changing of its I-V curves.（4） Replicated AD model rats by using D- galactose taking inject ion method after eye’s ball, recorded the neurotransmitters channel current and transmembrane voltage of AD model rats’ cerebral mRNA expressed in xenopus oocyte, and observed the influences of Shou’ erzhi medicated serum on transmembrane voltage and channel current.Results: （1） The qualitative detection of ligustrazine showed rabbits serum contained the drug ingredients of Shou’ erzhi herbs.（2） After separation, cultured hippocampus neurons cells grew well. Hippocampus neurons cells, cultured 12-15 days, attach wall completely, had greater cell volume, and was used to high resistance seals, and could observe synapse spontaneous activities remarkably in cell record.（3） Shou’ erzhi medicated serum could enhance the peak value of Na ion channel current effectively, accelerate Na ion channel inactivation; and also increase the peak value of K ion current obviously, reinforce K ion channel conductance; and inhibit the peak value of L- Ca current effectively, which has significant differences compared to blank group. （ p<0.05）（4） Shou’ erzhi medicated serum could strengthen transmembrane voltage and channel current of AD model rats’ cerebral mRNA expressed in xenopus oocyte, which has obviously discrepancies compared to blank group. （ p<0. 05）Conclusions: This research suggested that Shou’ erzhi capsule has apparent influences on the Na ion, K ion and L-Ca ion channel current and relative I-V curves of rats’ hippocampus neurons cells. Chinese herbal compound Shou’ erzhi could increase peak value of Na ion and K ion current (I_Na, I_k) apparently, and accelerate Na ion channel inactivation simultaneously. Then it also could reinforce K ion channel conductance, shorten depolarization time of hippocampus neurons, and generate strong amplitude action potential rapidly. Besides, it restrained hippocampus neurons cells depolarization continually and shortened its repolarization time. Shou’ erzhi inhibited the peak amplitude of L- Ca current effectively. Hence, it blocked Ca ion internal flow, and decreased Ca content in the cell to prevent apoptosis caused by Ca overload.By researching the effect of Shou’ erzhi on ion channel current expressed by AD model rats’ cerebral neurotransmitters, the results showed Shou’ erzhi can promote excitatory neurotransmitter release. Therefore, Shou’ erzhi could both stimulate the neurons cells and inhibit the excitatory neurotransmitter release excessively, through action mechanism mentioned above. Thereamong, accelerating Na ion channel inactivation and blocking Ca ion internal flow were the main functions to avoid cells damage by excitation toxic effect for neurotransmitter release excessively, which would prolong cells activities. Hence, Shou’ erzhi had definite functions to protect brain tissue and improve memory, aiming to prevent and treat Alzheimer’ s dementia.更多还原