【作者】 贺峰；

【导师】 谢俊霞；

【作者基本信息】 青岛大学 ， 生理学， 2004， 硕士

【摘要】 大量研究表明，β-淀粉样蛋白（Amyloid β-protein Aβ）能够促进电压门控性钙通道（voltage-gated calcium channels VGCC）开放，使Ca²⁺内流增多，引起胞内Ca²⁺超载，导致神经细胞损伤，凋亡，从而参与阿尔茨海默病（Alzheimer’s disease AD）的发病过程。流行病学研究发现，雌激素替代疗法可以显著降低AD的发病危险，改善AD病人的症状，延缓病程的进展；且许多基础研究表明，雌激素可通过多种途径对抗Aβ的毒性作用。另据文献报道，某些糖类及其衍生物具有一定的神经保护作用，在治疗神经变性疾病方面具有极大的潜质。本室的前期工作表明，分别应用天然的类雌激素药物—乌鸡白凤丸有效成分（Bak Foong Pills BFP）和海洋褐藻提取物—褐藻酸性寡糖HSH971，能够有效防止Aβ_1-40对大鼠肾上腺髓质嗜铬细胞瘤细胞（PC12细胞）的毒性作用。为探讨BFP和HSH971神经保护作用的可能机制，在本实验中，我们应用膜片钳全细胞记录技术观察了Aβ_1-40孵育对PC12细胞VGCC电流的影响，并在此基础上观察BFP、HSH971对此效应的干预作用。将PC12细胞分成空白对照组、Aβ_1-40组、BFP组、Aβ_1-40与BFP共孵育组、HSH971组、Aβ_1-40与HSH971共孵育组的VGCC的电流峰值，求得细胞的电流密度（电流值／膜电容），作为Ca²⁺内流的指标，比较各组间的差异。结果如下： 1．细胞在Aβ_1-401μM中孵育6h后，VGCC电流密度较空白对照组增大，差别有显著性。（P＜0.01） 2．BFP 100μg／ml与Aβ_1-401μM共同孵育细胞6h，VGCC电流密度与Aβ_1-40组中文摘要相比明显减小，差别具有高度统计学意义。（P<0.0]）HSH971 400组明显减小，以g/ml与A日、₄。l林M共同孵育细胞6h，VGCC电流密度较Ap，₄。 差别具有高度统计学意义。（P<0.0]）4.单独应用上述浓度的BFP和HSH971孵育细胞6h，VGCC电流密度较空白对照 组差别无显著性。（P>0.肠） 卜述结果提示，Apl₄。能够促进vGcc开放，c扩十内流增多，介导胞内钙超载，从而导致一系列生物学效应，造成细胞损伤;BFP能够显著抑制Ap，₄0所引起的VGCC电流增强效应，从而减少Ca2+内流，避免胞内钙离子超载，进而保护神经细胞，为AD的防治提供部分实验基础和理论依据。 本实验关于HSH971的研究显示，该寡糖也能够抑制A日】一4。诱导的VGCC介导的钙超载效应，初步证实了其部分神经保护作用。更多还原

【Abstract】 Previous studies have shown that amyloid beta-protein(A P ) can facilitate the opening of voltage- gated calcium channels(VGCC), leading to the increase of calcium inflow and overload of intracellular calcium which result in the damage and apoptosis of nerve cell. Therefore, A is involved in the pathogenesis of Alzheimer’s disease(AD). The epidemiology studies have shown that estrogen replacement therapy can decrease the risk of AD significantly, improve the symptom of AD patients and postpone the progress of AD. Furthermore, many studies have indicated that estrogen can inhibit the toxic effect of A P by many ways. Other studies indicate that some carbohydrate and their derivatives have a neuroprotective effect and have a great potential to treat the neurodegenerative diseases. Our previous experiments have shown that administration of native estrogen-like medicine, effective contraction of Bak Foong Pills(BFP), and ocean acidic oligosaccharide HSH971 can prevent the neurotoxicity of A P 1.40 on rat adrenal medulla Pheochromocytoma cells(PC12 cells) effectively. To understand the mechanism of BFP and HSH971 induced neuroprotective effect, we studied the effect of BFP and HSH971 on A induced facilitation of VGCC. PC 12 cells were divided into 6 groups: control group, A 1-40-treated group, BFP-treated group, A 1-40 +BFP-treated group, HSH971 -treated group and A 1-40 +HSH971 -treated group. Whole-cell patch-clamp recordings were performed in the present study to investigate the effects of different treatment on VGCC currents. Current density obtained by current-amplitude/membrane capacitance was used as a marker of Ca2+ inflax. The results were as follows:1. The VGCC current density of the cells incubated with A 1-40l M for 6h was significantly increased compared with that of the control group. (P<0.01)2. The VGCC current density of the cells co-incubated with A 1-40l M and BFP100 g/ml was significantly reduced compared with that of A -treated group.(P<0.01)3. Co-incubated with A 1-40 1MM and HSH971 400 g/ml for 6h, the VGCC current density was also significantly reduced compared with that of A -treated group. (P<0.01)4. There was no significant difference of the VGCC current density between control group and BFP or HSH971 treated group. (P>0.05)In summary, the present results suggested that A 1.40 can increase the VGCC currents of PC12 cell, which results in the increased Ca2+ influx leading to disruption of calcium homeostasis and activation of various biological effects causing cell damage. Furthermore, incubation with BFP can inhibit the effect of A P 1.40 on the VGCC currents obviously so that reduce the calcium influx, avoiding Ca2+ overload to provide neuroprotection. The results provides some experimental evidence for the prevention and treatment of Alzheimer’s disease.The present studies have also shown that HSH971 can reduce A 1-40 induced neurotoxicity of PC12 cells via inhibiting the effect of A 1-40 on the VGCC currents, which provide preliminary evidence for its neuroprotive effects.更多还原