【作者】 王珍琦；

【导师】 龚守良； 罗灿；

【作者基本信息】 吉林大学 ， 放射医学， 2004， 硕士

【摘要】 抗抑郁药的神经保护作用及其机制的研究日益引起国内外学者的关注，其慢性治疗可通过其神经保护作用加强海马神经元生成，促进神经元分枝，阻止抑郁症和应激时海马神经元的萎缩和坏死。目前，这一结论在动物和人体的研究中已被逐步证实。本研究采用体外原代培养的大鼠海马神经元，复制Glu损伤模型模拟抑郁症时的病理改变，观察4种抗抑郁药对经Glu损伤的神经元的存活、坏死及凋亡的影响；通过检测神经元细胞周期及酶活性的变化，初步探讨抗抑郁药的神经保护作用的机制。1 抗抑郁药神经保护作用的研究1.1 抗抑郁药对神经元存活的影响通过MTT比色法检测神经元增殖活性和杀伤活性。Glu的兴奋性神经毒性使神经元存活率降低约为正常的50% (p<0.05)；而抗抑郁药作用后，与Glu损伤组比较，均能不同程度地升高其存活率。其中，以olanzapine的存活率升高最为明显，而后依次为venlafaxine和quetiapine。提示venlafaxine、olanzapine和quatapine能够抵抗谷氨酸的兴奋性神经毒性，提高神经元的存活率，可能对培养的神经元具有直接的保护作用，有利于神经元的生长和存活。1.2 抗抑郁药对细胞膜通透性的影响通过检测谷氨酸诱导的神经元损伤及应用抗抑郁药物后乳酸脱氢酶（LDH）的释放，证实Glu损伤组LDH的释放急剧升高，为正常组的7.4倍；各种药物组与谷氨酸损伤组相比，均不同程度地减少LDH 的释放。同时，各药物在较低剂量时效果明显，而高剂量的作用不显著。各药物之间相比较，以olanzapine的作用最为明显。结果表明，所检测的几种药物均能抵抗谷氨酸诱导的神经元损伤，维持细胞膜的完整性，使LDH漏出减少，存活神经元数量增加，表现出较强的神经细胞保护作用。1.3 抗抑郁药对神经元坏死的影响 应用流式细胞术（FCM）检测坏死细胞比例的变化。谷氨酸的神经毒性使神经元坏死细胞比例升高3倍 (p<0.05)，而抗抑郁药组均不同程度降低<WP=64>Glu损伤所致坏死细胞比例升高。其中，venlafaxine在0.1~500 μmol/L时均降低坏死细胞比例，olanzapine组在50 μmol/L时作用最为显著 (p<0.01)，fluoxetine在10~100 μmol/L时作用显著 (p<0.01)。结果表明，抗抑郁药抑制了谷氨酸诱导的神经元坏死，具有神经保护作用。2 抗抑郁药神经保护作用机制的研究2.1 抗抑郁药对神经细胞周期的影响 通过FCM检测抗抑郁药物对Glu损伤的神经细胞周期和细胞增殖的影响。发现Glu诱导的神经元损伤G0/G1期细胞百分数增加，S期细胞下降，表明Glu抑制神经元的DNA合成，减少了细胞的增殖；而4种抗抑郁药的细胞周期的变化规律虽然不完全相同，但有一个共同点，即 G0/G1期细胞百分数不同程度地减少，而S期细胞增加，进入DNA合成期的细胞增加，促进细胞增殖，表明抗抑郁药可能是通过促进细胞增殖实现的。4种药物当中，以venlafaxine促进细胞增殖的作用最为明显。2.2 抗抑郁药对酶活性的影响Glu损伤后，SOD活性下降为正常组的12.3%，GSH的量下降为正常的9.2%，MDA水平急剧升高；而抗抑郁药应用后，SOD活性升高， GSH增加， MDA水平降低。提示，抗抑郁药可能通过升高SOD活性，增加GSH，降低MDA水平，抵抗抑郁和应激时HPA轴失调引起的自由基的神经毒害作用，增强了神经细胞抗氧化能力和抵抗自由基的能力，减弱Glu损伤，抑制氧化应激水平，提高清除自由基的能力，降低受自由基攻击的严重程度。本研究可能对于理解抗抑郁药的神经保护作用及阐明抗抑郁药的作用机理具有重要意义，也为指导临床合理用药提供了科学的实验依据；同时，有利于筛选有效、可靠的抗抑郁药物，以及为抗抑郁药治疗与海马神经元损害有关的神经精神疾病提供理论依据。更多还原

【Abstract】 The psychiacists have increasingly paid more attentions to the neuroprotective effect of antidepressant nowadays. It is reported that the chronic antidepressant administration enhances the neurogenesis and prompts the neuron sprouting in hippocampus by its neuroprotective effect, which can block or reverse the atrophy and death of hippocampal neurons in depression and stress. In many studies in human and animal, the conclusion has been verified to date. In this dissertation, the influence of four kinds of antidepressant drugs on rat primary cultured hippocampal neurons injured with glutamate to mimic the pathological changes induced by elevated glucocorticoid (GC) levels in depression or stress was studied. The present study is to observe the effects of four kinds of antidepressant drugs on neuron survival, death and apoptosis, and further explore the neuroprotective effect of antidepressants and its mechanism. The study on the neuroprotective effect of antidepressants The influence of antidepressants on neuron survival The neuron proliferation was detected with MTT. The results showed that the survival rate of neurons injured with the excitatory neurotoxicity of glutamate decreased to 48% of the control (p<0.05). However, the survival rates of neurons injured with glutamate increased in varying degree after the administration with four kinds of antidepressants as compared with those in the glutamate group. The effect of olanzapine was the most evident, followed by quetiapine and venlafaxine. The results demonstrate that these antidepressants can prevent the neurons in himppocampus from the damage induced with glutamate and elevate the neurons survival, which indicate that some antidepressants may have direct <WP=66>protective effect contributing to the proliferation and survival of neurons.1.2 The influence of antidepressants on the permeability of neuronal cell membrane The effects of antidepressants on the lactate dehydratase (LDH) release of neurons in hippocampus injured with glutamate were detected. The results showed that the LDH release of neurons injured with glutamate increased significantly, which was 7.4-fold of the control group. However, after administration with four kinds of antidepressants LDH release from neurons injured with glutamate decreased in varying degree as compared with that in the glutamate group. The effect of antidepressants on LDH release was decreased significantly in the lower concentrations of every antidepressant and was not in the higher concentrations. Meantime, olanzapine was more efficient than the other antidepressants. The results imply that the four kinds of antidepressants all can block the injury of neurons induced with glutamate by keeping the intactness of cell membrane, reducing LDH release and increasing the percentage of survival neurons to exhibit the neuroprotective effect of them on neurons.The influence of antidepressants on the neuron deathThe percentages of death neurons in hippocampus were detected with FCM in the experiment. The results showed that the percentages of neurons injured with glutamate increased approximately three-fold of the control. Nevertheless, the administration of four kinds of antidepressants reduced the percentages of neurons injured with glutamate in varying degree as compared with that in the glutamate group. The results demonstrate that four kinds of antidepressants can inhibit the death of neurons induced with glutamate by their neuroprotective effect.The study on the mechanism of the neuroprotective effect of antidepressants2.1 The influence of antidepressants on the cell cycle of neurons The changes in cell cycle and cell proliferation were detected with FCM in the present experiment. We found that the percentages of G0/G1 phase of neurons in happocampus injured with glutamate increased and those of S phase decreased. The results show that glutamate can inhibit the DNA synthesis and neuronal <WP=67>proliferation. In addition, though the change regulations of cell cycle in neurons were different after更多还原