PPARγ激动剂TGZ通过JNK通路促进海马神经元轴突生长机制研究

发布时间：2018-02-08 19:28

  本文关键词： TGZ PPARγ JNK通路 海马神经元 轴突生长 神经退行性疾病　出处：《承德医学院》2014年硕士论文　论文类型：学位论文

【摘要】：海马在中枢神经系统发挥重要作用，与理解、想象等功能有着密切的联系，许多神经系统退行性疾病如阿尔茨海默病（AD）、帕金森病（PD）、亨廷顿舞蹈症（HD）、肌萎缩性脊髓侧索硬化症(ALS)等都与该部位的病变有关[1、2]。根据目前的研究，一般认为神经功能退化可能与神经元的丢失有关，但越来越多的证据表明，在正常老化过程中，神经元的数量并没有发生明显的变化，轴突变短或缺失而导致突触功能异常和神经细胞的死亡才是神经退行性疾病的基本病理特征[3、4]。 过氧化物酶体增殖物激活受体(PPARs)通过基因转录水平的调控来发挥其作用。目前的研究表明, PPARs可以保护神经细胞,在治疗神经退行性疾病方面具有潜在性的应用价值[5]。PPARs包括PPARα、PPARβ、PPARγ三种亚型，PPARγ以其与代谢性疾病的特殊作用成为研究热点，PPARγ只有与相应的配体结合才能发挥生理学效应，曲格列酮（TGZ）就是一种PPARγ强效激活剂，被广泛用于糖尿病的临床治疗，但是在神经系统疾病方面的应用还比较少[6]。通过研究发现，，用PPARγ受体激动剂处理嗜铬细胞瘤细胞（PC12）后，可以明显促进其突触的延长，并且这种促进作用与MAPK-JNK通路的活化相关，但PPARγ通路是否会对神经元轴突的生长产生影响尚未见报道[7]。本课题就是以神经元种类中最具代表性的海马神经元作为研究对象，通过PPARγ受体激动剂及拮抗剂的处理，探究PPARγ在海马神经元轴突延长过程中作用及其机制。 目的： PPARγ蛋白激活剂TGZ可以促进海马神经元轴突的生长，其机制是JNK通路介导TGZ发挥促轴突生长作用。 方法： 分离获得纯度达到90%以上的胚胎源性的小鼠海马神经元，TGZ和GW9662处理细胞，随机分为control组、TGZ组、GW组及TGZGW组四组，通过western blot验证TGZ和GW9662对PPARγ表达的调节作用及对JNK的活化作用。TGZ、GW9662、SP600125分不同组别处理细胞，通过细胞免疫荧光染色定量分析细胞轴突生长的情况。 结果： 经anti-tau染色鉴定得出，分离培养的原代海马神经元纯度在90%以上；western blot结果证明TGZ和GW9662可以有效调节PPARγ蛋白的表达水平，而且TGZ和GW9662可以促进JNK蛋白的磷酸化水平；TGZ可以促进轴突的延长（P0.05），且作用在72h效果最显著，GW9662可以抑制TGZ的促轴突作用（P0.05），验证了TGZ可以有效活化PPARγ蛋白，应用JNK通路特异性抑制剂SP600125后，TGZ的促轴突作用受到了明显抑制（P0.05），这就证明TGZ的促轴突作用是建立在JNK通路活化基础之上的。 结论： TGZ可以促进海马神经元轴突的延长，这种作用是通过活化PPARγ实现的，而且这种作用机制是由JNK通路介导的。
[Abstract]:The hippocampus plays an important role in the central nervous system and is closely related to the functions of understanding, imagination, etc. Many neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HDD), muscular atrophic lateral sclerosis (ALS), and so on, are associated with this site [1] .According to current studies, It is generally believed that the degeneration of neural function may be related to the loss of neurons, but there is increasing evidence that the number of neurons does not change significantly during normal aging. Synaptic dysfunction and neuronal death due to axonal shortening or deletion are the basic pathological features of neurodegenerative diseases. Peroxisome proliferator activated receptor (PPAR) plays its role through regulation of gene transcription level. Current studies have shown that PPARs protects nerve cells. The potential application value of PPARs in the treatment of neurodegenerative diseases [5] .PPARs include three subtypes of PPAR 伪 -PPAR- 尾 -PPAR- 纬. PPAR- 纬 has become a research hotspot because of its special role in metabolic diseases. PPAR- 纬 can play a physiological effect only when it binds with corresponding ligands. Trioglitazone (TGZ) is a powerful activator of PPAR 纬, which is widely used in the clinical treatment of diabetes, but it is rarely used in nervous system diseases [6]. It was found that PPAR 纬 receptor agonist was used to treat pheochromocytoma cell line PC12). It can obviously promote the synaptic prolongation of its synapses, and this effect is related to the activation of the MAPK-JNK pathway. However, it has not been reported whether the PPAR 纬 pathway will affect the growth of neuronal axons [7]. This study focuses on hippocampal neurons, which are the most representative of neuronal types, and are treated by PPAR 纬 receptor agonists and antagonists. To explore the role and mechanism of PPAR 纬 in axonal extension of hippocampal neurons. Objective:. PPAR 纬 protein activator TGZ can promote axonal growth of hippocampal neurons, and its mechanism is that JNK pathway mediates TGZ to promote axon growth. Methods:. Mouse hippocampal neurons with purity of more than 90% were isolated and treated with TGZ and GW9662. They were randomly divided into four groups: control group, TGZ group and TGZGW group. The effects of TGZ and GW9662 on the expression of PPAR 纬 and the activation of JNK were verified by western blot. TGZ GW9662 and SP600125 were divided into different groups, and the cell axon growth was quantitatively analyzed by cell immunofluorescence staining. Results:. The results of anti-tau staining showed that the purity of cultured primary hippocampal neurons was more than 90%. The results showed that TGZ and GW9662 could effectively regulate the expression of PPAR 纬 protein. Moreover, TGZ and GW9662 could promote the phosphorylation level of JNK protein. TGZ could promote the prolongation of axon (P0.05N), and GW9662 could inhibit the axon action of TGZ (P0.05N) at 72 h, which proved that TGZ could effectively activate PPAR 纬 protein. The axon stimulating effect of TGZ was significantly inhibited by SP600125, a specific inhibitor of JNK pathway, which suggested that the axon stimulating effect of TGZ was based on the activation of JNK pathway. Conclusion:. TGZ can promote axonal lengthening of hippocampal neurons by activating PPAR 纬, and this mechanism is mediated by JNK pathway.
【学位授予单位】：承德医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R741


本文编号：1496089