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丙戊酸钠对PD细胞模型的保护作用研究

发布时间:2018-08-31 12:16
【摘要】:帕金森病(Parkinson’s disease, PD)是一种常见的进展缓慢的神经系统退行性疾病,到目前为止,PD还不能完全治愈,探寻该病更好的治疗策略是亟待解决的难题。丙戊酸钠(valproate, VPA),在临床上用于治疗双相精神障碍和癫痫,也可用于缓解神经退行性疾病的症状;现有研究表明,VPA在体内外都具有神经保护和神经营养作用,促进神经细胞轴突生长,抑制神经细胞凋亡。成束和延伸蛋白(fasciculation andelongation protein zeta1, FEZ1)在神经细胞发育、胞内运输、细胞凋亡和神经递质的释放中起到重要的作用,参与中枢神经系统发育及轴突的生长和成束。肾上腺嗜铬细胞瘤PC12,可以合成分泌多巴胺,是多巴胺能神经元体外培养的细胞模型,常被用来建立体外PD细胞模型。 目的:研究VPA对PC12细胞增殖、迁移及对FEZ1表达的影响;建立PD细胞模型,探讨VPA对6-OHDA诱导的PC12细胞损伤的神经保护和抗氧化作用。 方法:(1)采用0.5、0.75、1.0mM的VPA分别处理PC12细胞24h,观察细胞的形态学变化和细胞迁移作用的影响,以MTT法检测VPA对细胞增殖作用的影响,采用real-time PCR与Western blot检测FEZ1的表达变化。(2)建立PD细胞模型,将细胞分组:正常对照组、6-OHDA组、VPA(0.5、0.75、1.0mM)+6-OHDA组;观察各组细胞的动态变化,以MTT法检测各组细胞的增殖情况,采用real-time PCR与Western blot检测FEZ1及凋亡相关基因的表达变化,检测各组细胞的SOD活性和MDA含量。 结果:(1) MTT及细胞划痕结果显示,VPA能促进PC12细胞增殖与迁移,并呈剂量依赖性;real-time PCR与Western blot结果显示,VPA能促进FEZ1mRNA和蛋白的表达,并呈剂量依赖性。(2)用6-OHDA处理PC12细胞制备PD细胞模型,PC12细胞增殖受到抑制;而用VPA预处理24h后,再以6-OHDA处理24h,细胞凋亡状况有所缓解,细胞皱缩数量降低,突起伸长,相互接触的细胞数量增多。real-time PCR与Western blot检测各处理组FEZ1、Bcl-2和Bax的表达结果显示:6-OHDA组与正常对照组相比,,FEZ1和Bcl-2表达下降,Bax表达上升;VPA+6-OHDA组与6-OHDA组相比,FEZ1和Bcl-2表达上升,Bax表达下降。SOD活性和MDA含量的检测结果显示:6-OHDA组与正常对照组相比SOD活性下降,MDA含量上升,VPA+6-OHDA组与6-OHDA组相比,SOD活性增加,MDA含量下降。 结论:(1) VPA在实验浓度范围内能促进PC12细胞增殖、迁移,提高FEZ1mRNA及其蛋白表达,呈剂量依赖性。(2) VPA使6-OHDA诱导的PD细胞模型FEZ1和Bcl-2表达上调、Bax表达下调,提高细胞的抗氧化水平,减轻氧化应激对细胞的损伤,进而减少细胞凋亡和促进细胞增殖,发挥神经保护作用。
[Abstract]:Parkinson's disease (Parkinson's disease, PD) is a common neurodegenerative disease with slow progression. So far, PD can not be completely cured. Sodium valproate (valproate, VPA), is used clinically to treat bipolar disorder and epilepsy, but also to relieve the symptoms of neurodegenerative diseases. Promote neuronal axon growth and inhibit neuronal apoptosis. Bundles and extension proteins (fasciculation andelongation protein zeta1, FEZ1) play an important role in the development of nerve cells, intracellular transport, apoptosis and release of neurotransmitters, and participate in the development of the central nervous system and the growth and bunching of axons. Adrenal pheochromocytoma (PC12,), which can synthesize and secrete dopamine, is a cell model of dopaminergic neurons cultured in vitro. It is often used to establish PD cell model in vitro. Aim: to study the effects of VPA on the proliferation, migration and FEZ1 expression of PC12 cells, and to establish a PD cell model to investigate the neuroprotective and antioxidant effects of VPA on 6-OHDA induced PC12 cell injury. Methods: (1) PC12 cells were treated with 0.5-0.75 mm VPA for 24 h. The morphological changes and the effects of cell migration were observed. The effect of VPA on cell proliferation was detected by MTT assay. Real-time PCR and Western blot were used to detect the expression of FEZ1. (2) the PD cell model was established, and the cells were divided into normal control group and 6-OHDA group. The dynamic changes of the cells were observed in the 6-OHDA group, and the proliferation of the cells in each group was detected by MTT method. Real-time PCR and Western blot were used to detect the expression of FEZ1 and apoptosis-related genes, and the activity of SOD and the content of MDA were detected. Results: (1) the results of MTT and cell scratch showed that VPA could promote the proliferation and migration of PC12 cells. The results of real-time PCR and Western blot showed that VPA could promote the expression of FEZ1mRNA and protein in a dose-dependent manner. In a dose-dependent manner. (2) the proliferation of PD cells was inhibited by PC12 cells treated with 6-OHDA, and then treated with 6-OHDA for 24 h after pretreatment with VPA for 24 h, the cell apoptosis was alleviated, the number of cell shrinkage was decreased, and the protrusions were elongated. The expression of FEZ1,Bcl-2 and Bax in each treatment group was detected by real-time PCR and Western blot. The results showed that the expression of FEZ1 and Bcl-2 decreased in the control group compared with the control group. The expression of FEZ1 and Bcl-2 in VPA 6-OHDA group was higher than that in 6-OHDA group. The results showed that the activity of SOD in VPA 6-OHDA group was lower than that in control group. The content of SOD in VPA 6-OHDA group was higher than that in 6-OHDA group, and the content of MDA in VPA 6-OHDA group was higher than that in 6-OHDA group. Conclusion: (1) VPA can promote the proliferation and migration of PC12 cells and increase the expression of FEZ1mRNA and its protein in a dose-dependent manner. (2) VPA can up-regulate the expression of FEZ1 and Bcl-2 in PD cell model induced by 6-OHDA and increase the level of anti-oxidation. It can reduce cell apoptosis, promote cell proliferation and play a neuroprotective role.
【学位授予单位】:苏州大学
【学位级别】:硕士
【学位授予年份】:2014
【分类号】:R742.5

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