5-LO表达与铝盐致海马神经元损伤的关系研究

发布时间：2018-04-05 12:03

本文选题：神经元损伤　切入点：铝盐　出处：《重庆医科大学》2010年硕士论文

【摘要】： 目的:建立氯化铝致原代培养大鼠海马神经元损伤模型,探讨5-LO的表达与铝盐致海马神经元损伤的关系。方法: (1)新生24h内SD大鼠海马神经元原代培养,7天后用免疫组化鉴定纯度;给予氯化铝(Al3+ 200μmol·L-1)建立原代培养大鼠海马神经元损伤模型。 (2)以HE染色及荧光显微镜观察海马神经元病理形态变化,采用生化方法检测MTT值、LDH漏出率、SOD活性和MDA含量,分别用RT-PCR和Western Blotting方法检测5-LO mRNA和蛋白表达。 (3)5-LO过表达腺病毒转染原代培养海马神经元,观察5-LO过表达神经元的变化,以及5-LO过表达对铝盐致海马神经元损伤的易感性。 (4)用咖啡酸抑制5-LO活性,RNA干扰抑制5-LO表达,观察其对铝盐致海马神经元损伤的作用。结果: (1)神经元纯度超过95%。铝负荷致原代培养海马神经元数目明显减少,神经元突起减少变短,活力降低,LDH漏出率明显增加,SOD活性降低,MDA含量升高,5-LO mRNA和蛋白表达增加。 (2)与空载腺病毒转染组相比,单纯5-LO过表达神经元的数目、形态,细胞活力,LDH漏出率,SOD活性,MDA含量无明显变化,5-LO mRNA和5-LO蛋白表达增加。与铝盐加空载腺病毒转染组相比,5-LO过表达神经元给予铝盐负荷,神经元数目减少更为明显,细胞碎片增加,细胞活力和SOD活性显著降低,LDH漏出率和MDA含量显著增加,5-LO mRNA和5-LO蛋白表达也明显增加。 (3)咖啡酸给予能剂量依赖性地减轻铝负荷/和5-LO腺病毒过表达海马神经元损伤,提高神经元存活力,降低LDH漏出率,明显阻遏铝负荷神经元SOD活性的降低和MDA含量的升高,能下调5-LO mRNA和蛋白的表达。 (4)5-LO RNA干扰能明显减轻铝负荷所致的海马神经元损伤,提高神经元的存活力,降低LDH的漏出率,明显阻遏铝负荷神经元SOD活性的降低和MDA含量的升高,明显降低5-LO mRNA和蛋白的表达。结论: (1)神经元单纯过表达5-LO并不引起明显的细胞损伤,但能增加神经元对铝负荷损伤的易感性。 (2)抑制5-LO活性、下调5-LO表达对铝负荷致海马神经元损伤有明显的保护作用。
[Abstract]:Aim: to establish the model of primary cultured rat hippocampal neuron injury induced by aluminum chloride, and to investigate the relationship between the expression of 5-LO and the damage of hippocampal neurons induced by aluminum salt.Methods:(1) the primary cultured hippocampal neurons of SD rats within 24 hours were cultured for 7 days, the purity of hippocampal neurons was identified by immunohistochemistry, and the injury model of hippocampal neurons in primary cultured rats was established by Al 3 200 渭 mol L -1).(2) the histopathological changes of hippocampal neurons were observed by HE staining and fluorescence microscope. Sod activity and MDA content were detected by biochemical method and 5-LO mRNA and protein expression were detected by RT-PCR and Western Blotting respectively.The changes of 5-LO overexpression neurons and the susceptibility of 5-LO overexpression to the damage of hippocampal neurons induced by aluminum salt were observed in primary cultured hippocampal neurons transfected with adenovirus.(4) the inhibitory effect of caffeic acid on the expression of 5-LO in hippocampal neurons induced by aluminum salt was observed.Results:The purity of neurons was more than 95%.The number of primary cultured hippocampal neurons was significantly decreased, the neuronal process was decreased, the leakage rate of LDH was decreased, the activity of SOD was increased, the content of malondialdehyde (MDA) was increased and the expression of 5-LO mRNA and protein was increased in primary cultured hippocampal neurons.(2) compared with the control group, the number and morphology of 5-LO overexpression neurons, the leakage rate of 5-LO activity and the content of 5-LO protein were not significantly increased in the control group compared with those in the non-loaded adenovirus transfection group, and the expression of 5-LO mRNA and 5-LO protein were not significantly increased.Compared with the aluminum-salt + no-load adenovirus transfection group, the number of neurons decreased and the cell fragments increased when the overexpression neurons of 5-LO were loaded with aluminum salt.Cell viability and SOD activity significantly decreased the leakage rate and the content of MDA significantly increased the expression of 5-LO mRNA and 5-LO protein.(3) caffeic acid could attenuate the damage of hippocampal neurons under aluminum load / 5-LO overexpression in a dose-dependent manner, increase the viability of neurons, decrease the leakage rate of LDH, and significantly inhibit the decrease of SOD activity and the increase of MDA content in aluminum-loaded neurons.It can down-regulate the expression of 5-LO mRNA and protein.The interference of 5-LO RNA could significantly reduce the damage of hippocampal neurons induced by aluminum load, increase the viability of neurons, decrease the leakage rate of LDH, and inhibit the decrease of SOD activity and the increase of MDA content in the neurons under aluminum loading.The expression of 5-LO mRNA and protein was significantly decreased.Conclusion:1) single overexpression of 5-LO in neurons did not cause obvious cell damage, but increased the susceptibility of neurons to aluminum load damage.2) inhibition of 5-LO activity and down-regulation of 5-LO expression have obvious protective effects on hippocampal neuron injury induced by aluminum load.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R363

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