本文選題：帕金森病　切入點(diǎn)：Olanzapine　出處：《蘇州大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：目的：研究奧氮平（Olanzapine, OLA）對(duì)rotenone誘導(dǎo)多巴胺（dopamine, DA）能神經(jīng)元損傷的保護(hù)作用及其可能的機(jī)制。 方法：（1）細(xì)胞實(shí)驗(yàn)：首先檢測(cè)olanzapine對(duì)PC12細(xì)胞的毒性作用，，采用Western blot檢測(cè)olanzapine對(duì)p-AMPK以及自噬相關(guān)蛋白LC3，p62，p70-s6k的影響，免疫熒光染色檢測(cè)LC3的變化。采用0.5μM rotenone處理PC12細(xì)胞24h建立PD細(xì)胞模型，治療組在rotenone給藥前24h加入olanzapine（20μM）預(yù)處理，實(shí)驗(yàn)組以終濃度為2.5μM的AMPK選擇性抑制劑compound C預(yù)處理PC12細(xì)胞3h后再加入olanzapine及rotenone。采用CCK-8法檢測(cè)細(xì)胞活性；Hoechst33258染色法觀察細(xì)胞核形態(tài)變化；Western blot法觀察凋亡蛋白PARP及α-synuclein的表達(dá)情況。（2）動(dòng)物實(shí)驗(yàn)：雄性Lewis大鼠52只隨機(jī)分為4組：rotenone注射組的大鼠于頸背部皮下注射rotenone（1.5mg/kg/day）8周，治療組在每天灌胃0.9mg/kg olanzapine，八周后觀察其行為學(xué)變化，處死動(dòng)物取腦、切片，免疫組化染色觀察酪氨酸羥化酶（TH）、LC3及α-synuclein的表達(dá)。 結(jié)果：Western blot結(jié)果顯示olanzapine能夠顯著激活A(yù)MPK并上調(diào)LC3II的表達(dá)，下調(diào)p62以及p70-s6k的水平，表明olanzapine能夠激活自噬。CCK-8及Hoechst33258結(jié)果提示olanzapine能夠減輕rotenone誘導(dǎo)的PC12細(xì)胞的凋亡，降低由rotenone引起的α-synuclein和PARP的增高。當(dāng)抑制AMPK以后，自噬的誘導(dǎo)被阻礙并且保護(hù)作用消失。體內(nèi)實(shí)驗(yàn)結(jié)果顯示，olanzapine能夠上調(diào)大鼠腦內(nèi)LC3表達(dá)。治療組的大鼠的行為計(jì)分顯著低于模型組，治療組的黑質(zhì)致密部TH陽(yáng)性細(xì)胞數(shù)較模型組明顯增加，且殘存的TH陽(yáng)性細(xì)胞內(nèi)的α-synuclein顯著減少。 結(jié)論：olanzapine可以減輕rotenone對(duì)多巴胺能神經(jīng)元的損傷，這種保護(hù)作用可能是通過激活A(yù)MPK通路，上調(diào)自噬實(shí)現(xiàn)的。
[Abstract]:Objective: To study the protective effect of Olanzapine (OLA) on rotenone induced dopamine (dopamine, DA) neuron injury and its possible mechanism.
Methods: (1) cell toxicity experiment: first detection of olanzapine in PC12 cells, using Western blot and p-AMPK olanzapine detection of autophagy related protein LC3, p62, p70-s6k, LC3 changes were detected by immunofluorescence staining. Using 0.5 M rotenone PC12 cell 24h establishment of PD cell model, the treatment group in rotenone administration of 24h before joining olanzapine (20 M) pretreatment, the experimental group with a final concentration of 2.5 M AMPK selective inhibitor of compound C pretreatment of PC12 cells after 3H olanzapine and rotenone. CCK-8 was used to detect cell activity; to observe morphological changes of nucleus Hoechst33258 staining; Western blot method was used to observe the expression of apoptosis protein PARP alpha and -synuclein. (2) animal experiment: 52 male Lewis rats were randomly divided into 4 groups: Group rotenone injection the rats in the subcutaneous injection of rotenone (1.5mg/kg/day) for 8 weeks, treatment The rats in each group were fed 0.9mg/kg olanzapine for eight days, then their behavioral changes were observed. The animals were killed and the brains were sectioning. Immunohistochemical staining was used to observe the expression of tyrosine hydroxylase (TH), LC3 and -synuclein.
Results: Western blot showed that olanzapine could activate the expression of AMPK and upregulation of LC3II, downregulation of p62 and p70-s6k, indicated that olanzapine could activate autophagy in.CCK-8 and Hoechst33258 indicated olanzapine can reduce the rotenone induced apoptosis of PC12 cells, decrease caused by rotenone alpha -synuclein and PARP. When the inhibition of autophagy after AMPK blocked and induced protective effect disappeared. In vivo results showed that olanzapine can upregulate the expression of LC3 in brain of rats. Rats in the treatment group behavior score was significantly lower than the model group, the number of TH positive cells in substantia nigra in the treatment group were significantly increased compared with the model group, the alpha -synuclein TH positive cells and the remnants of the significant reduced.
Conclusion: Olanzapine can reduce the damage of rotenone to dopaminergic neurons. This protective effect may be achieved by activating the AMPK pathway to increase the realization of autophagy.

【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

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 陳忻;張楠;趙暉;穆陽(yáng);;魚藤酮致帕金森病大鼠行為學(xué)與黑質(zhì)病理?yè)p傷的關(guān)系[J];中國(guó)神經(jīng)精神疾病雜志;2008年04期

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