本文關(guān)鍵詞： TGZ PPARγ JNK通路 海馬神經(jīng)元 軸突生長 神經(jīng)退行性疾病　出處：《承德醫(yī)學院》2014年碩士論文　論文類型：學位論文

【摘要】：海馬在中樞神經(jīng)系統(tǒng)發(fā)揮重要作用，與理解、想象等功能有著密切的聯(lián)系，許多神經(jīng)系統(tǒng)退行性疾病如阿爾茨海默病（AD）、帕金森�。≒D）、亨廷頓舞蹈癥（HD）、肌萎縮性脊髓側(cè)索硬化癥(ALS)等都與該部位的病變有關(guān)[1、2]。根據(jù)目前的研究，一般認為神經(jīng)功能退化可能與神經(jīng)元的丟失有關(guān)，但越來越多的證據(jù)表明，在正常老化過程中，神經(jīng)元的數(shù)量并沒有發(fā)生明顯的變化，軸突變短或缺失而導致突觸功能異常和神經(jīng)細胞的死亡才是神經(jīng)退行性疾病的基本病理特征[3、4]。 過氧化物酶體增殖物激活受體(PPARs)通過基因轉(zhuǎn)錄水平的調(diào)控來發(fā)揮其作用。目前的研究表明, PPARs可以保護神經(jīng)細胞,在治療神經(jīng)退行性疾病方面具有潛在性的應用價值[5]。PPARs包括PPARα、PPARβ、PPARγ三種亞型，PPARγ以其與代謝性疾病的特殊作用成為研究熱點，PPARγ只有與相應的配體結(jié)合才能發(fā)揮生理學效應，曲格列酮（TGZ）就是一種PPARγ強效激活劑，被廣泛用于糖尿病的臨床治療，但是在神經(jīng)系統(tǒng)疾病方面的應用還比較少[6]。通過研究發(fā)現(xiàn)，，用PPARγ受體激動劑處理嗜鉻細胞瘤細胞（PC12）后，可以明顯促進其突觸的延長，并且這種促進作用與MAPK-JNK通路的活化相關(guān)，但PPARγ通路是否會對神經(jīng)元軸突的生長產(chǎn)生影響尚未見報道[7]。本課題就是以神經(jīng)元種類中最具代表性的海馬神經(jīng)元作為研究對象，通過PPARγ受體激動劑及拮抗劑的處理，探究PPARγ在海馬神經(jīng)元軸突延長過程中作用及其機制。 目的： PPARγ蛋白激活劑TGZ可以促進海馬神經(jīng)元軸突的生長，其機制是JNK通路介導TGZ發(fā)揮促軸突生長作用。 方法： 分離獲得純度達到90%以上的胚胎源性的小鼠海馬神經(jīng)元，TGZ和GW9662處理細胞，隨機分為control組、TGZ組、GW組及TGZGW組四組，通過western blot驗證TGZ和GW9662對PPARγ表達的調(diào)節(jié)作用及對JNK的活化作用。TGZ、GW9662、SP600125分不同組別處理細胞，通過細胞免疫熒光染色定量分析細胞軸突生長的情況。 結(jié)果： 經(jīng)anti-tau染色鑒定得出，分離培養(yǎng)的原代海馬神經(jīng)元純度在90%以上；western blot結(jié)果證明TGZ和GW9662可以有效調(diào)節(jié)PPARγ蛋白的表達水平，而且TGZ和GW9662可以促進JNK蛋白的磷酸化水平；TGZ可以促進軸突的延長（P0.05），且作用在72h效果最顯著，GW9662可以抑制TGZ的促軸突作用（P0.05），驗證了TGZ可以有效活化PPARγ蛋白，應用JNK通路特異性抑制劑SP600125后，TGZ的促軸突作用受到了明顯抑制（P0.05），這就證明TGZ的促軸突作用是建立在JNK通路活化基礎(chǔ)之上的。 結(jié)論： TGZ可以促進海馬神經(jīng)元軸突的延長，這種作用是通過活化PPARγ實現(xiàn)的，而且這種作用機制是由JNK通路介導的。
[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 偽 -PPAR- 尾 -PPAR- 緯. PPAR- 緯 has become a research hotspot because of its special role in metabolic diseases. PPAR- 緯 can play a physiological effect only when it binds with corresponding ligands. Trioglitazone (TGZ) is a powerful activator of PPAR 緯, 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 緯 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 緯 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 緯 receptor agonists and antagonists. To explore the role and mechanism of PPAR 緯 in axonal extension of hippocampal neurons. Objective:. PPAR 緯 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 緯 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 緯 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 緯 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 緯, and this mechanism is mediated by JNK pathway.
【學位授予單位】：承德醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R741


本文編號：1496089