【摘要】：帕金森病(Parkinson’s disease, PD)是一種常見的進(jìn)展緩慢的神經(jīng)系統(tǒng)退行性疾病，到目前為止，PD還不能完全治愈，探尋該病更好的治療策略是亟待解決的難題。丙戊酸鈉(valproate, VPA)，在臨床上用于治療雙相精神障礙和癲癇,也可用于緩解神經(jīng)退行性疾病的癥狀；現(xiàn)有研究表明，VPA在體內(nèi)外都具有神經(jīng)保護(hù)和神經(jīng)營養(yǎng)作用，促進(jìn)神經(jīng)細(xì)胞軸突生長，抑制神經(jīng)細(xì)胞凋亡。成束和延伸蛋白(fasciculation andelongation protein zeta1, FEZ1)在神經(jīng)細(xì)胞發(fā)育、胞內(nèi)運(yùn)輸、細(xì)胞凋亡和神經(jīng)遞質(zhì)的釋放中起到重要的作用，參與中樞神經(jīng)系統(tǒng)發(fā)育及軸突的生長和成束。腎上腺嗜鉻細(xì)胞瘤PC12，可以合成分泌多巴胺，是多巴胺能神經(jīng)元體外培養(yǎng)的細(xì)胞模型，常被用來建立體外PD細(xì)胞模型。 目的：研究VPA對(duì)PC12細(xì)胞增殖、遷移及對(duì)FEZ1表達(dá)的影響；建立PD細(xì)胞模型，探討VPA對(duì)6-OHDA誘導(dǎo)的PC12細(xì)胞損傷的神經(jīng)保護(hù)和抗氧化作用。 方法：(1)采用0.5、0.75、1.0mM的VPA分別處理PC12細(xì)胞24h，觀察細(xì)胞的形態(tài)學(xué)變化和細(xì)胞遷移作用的影響，以MTT法檢測(cè)VPA對(duì)細(xì)胞增殖作用的影響，采用real-time PCR與Western blot檢測(cè)FEZ1的表達(dá)變化。(2)建立PD細(xì)胞模型，將細(xì)胞分組：正常對(duì)照組、6-OHDA組、VPA(0.5、0.75、1.0mM)+6-OHDA組；觀察各組細(xì)胞的動(dòng)態(tài)變化，以MTT法檢測(cè)各組細(xì)胞的增殖情況，采用real-time PCR與Western blot檢測(cè)FEZ1及凋亡相關(guān)基因的表達(dá)變化，檢測(cè)各組細(xì)胞的SOD活性和MDA含量。 結(jié)果：(1) MTT及細(xì)胞劃痕結(jié)果顯示，VPA能促進(jìn)PC12細(xì)胞增殖與遷移，并呈劑量依賴性；real-time PCR與Western blot結(jié)果顯示，VPA能促進(jìn)FEZ1mRNA和蛋白的表達(dá)，并呈劑量依賴性。(2)用6-OHDA處理PC12細(xì)胞制備PD細(xì)胞模型，PC12細(xì)胞增殖受到抑制；而用VPA預(yù)處理24h后，再以6-OHDA處理24h，細(xì)胞凋亡狀況有所緩解，細(xì)胞皺縮數(shù)量降低，突起伸長，相互接觸的細(xì)胞數(shù)量增多。real-time PCR與Western blot檢測(cè)各處理組FEZ1、Bcl-2和Bax的表達(dá)結(jié)果顯示：6-OHDA組與正常對(duì)照組相比，，F(xiàn)EZ1和Bcl-2表達(dá)下降，Bax表達(dá)上升；VPA+6-OHDA組與6-OHDA組相比，F(xiàn)EZ1和Bcl-2表達(dá)上升，Bax表達(dá)下降。SOD活性和MDA含量的檢測(cè)結(jié)果顯示：6-OHDA組與正常對(duì)照組相比SOD活性下降，MDA含量上升，VPA+6-OHDA組與6-OHDA組相比，SOD活性增加，MDA含量下降。 結(jié)論：(1) VPA在實(shí)驗(yàn)濃度范圍內(nèi)能促進(jìn)PC12細(xì)胞增殖、遷移，提高FEZ1mRNA及其蛋白表達(dá)，呈劑量依賴性。(2) VPA使6-OHDA誘導(dǎo)的PD細(xì)胞模型FEZ1和Bcl-2表達(dá)上調(diào)、Bax表達(dá)下調(diào)，提高細(xì)胞的抗氧化水平，減輕氧化應(yīng)激對(duì)細(xì)胞的損傷，進(jìn)而減少細(xì)胞凋亡和促進(jìn)細(xì)胞增殖，發(fā)揮神經(jīng)保護(hù)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.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R742.5

【共引文獻(xiàn)】

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