【摘要】：帕金森病(Parkinson's disease, PD)是一種常見(jiàn)的神經(jīng)系統(tǒng)退行性疾病。隨著研究的深入,PD不再認(rèn)為是僅僅在黑質(zhì)—紋狀體系統(tǒng)發(fā)生了病變,而是一個(gè)影響周圍神經(jīng)系統(tǒng)和中樞神經(jīng)系統(tǒng)多個(gè)核團(tuán)的疾病。PD患者的認(rèn)知能力受損的發(fā)生率高達(dá)84%。 迄今為止,PD的病因仍未完全闡明,盡管遺傳和環(huán)境因素都起作用,但是90%以上的PD病人是散發(fā)性的。流行病學(xué)研究提示,環(huán)境因素與PD發(fā)病密切相關(guān),長(zhǎng)期接觸農(nóng)藥的人群,PD的發(fā)病率較高。魚藤酮是一種常用的除草劑和殺蟲劑,能夠通過(guò)血腦屏障,通過(guò)抑制線粒體呼吸鏈復(fù)合物I的活性,從而導(dǎo)致黑質(zhì)多巴胺能神經(jīng)元的能量合成障礙,進(jìn)而引起神經(jīng)元的死亡。因此,魚藤酮被用于PD模型的制備。與其他的PD動(dòng)物模型相比,魚藤酮制備的PD模型具有明顯的優(yōu)勢(shì),它能夠模擬PD的病理性特征變化,包括黑質(zhì)多巴胺能神經(jīng)元數(shù)目的降低和殘存神經(jīng)元內(nèi)的alpha-突觸核蛋白的聚集。 目前,對(duì)魚藤酮制備的PD模型的研究,多集中在黑質(zhì)—紋狀體系統(tǒng)的功能改變上,對(duì)其空間學(xué)習(xí)和記憶能力的改變尚未見(jiàn)報(bào)道。為了觀察PD中認(rèn)知功能的改變并初步探討其機(jī)制,本實(shí)驗(yàn)選用12月齡的C57BL/6小鼠進(jìn)行魚藤酮灌胃處理,動(dòng)物分為對(duì)照組,魚藤酮處理3月組和處理1月組,應(yīng)用免疫熒光和硫酸素T染色法觀察黑質(zhì)多巴胺能神經(jīng)元數(shù)目的改變和神經(jīng)元內(nèi)alpha-突觸核蛋白聚集的情況,應(yīng)用轉(zhuǎn)棒法觀察小鼠運(yùn)動(dòng)協(xié)調(diào)能力的改變,應(yīng)用Morris水迷宮評(píng)價(jià)小鼠空間學(xué)習(xí)與記憶能力的改變,同時(shí)通過(guò)免疫熒光雙標(biāo)的方法標(biāo)記海馬神經(jīng)元的增殖細(xì)胞核抗原和微管相關(guān)蛋白,反映海馬未成熟神經(jīng)元的變化。研究結(jié)果如下： 1.在魚藤酮處理3月組,小鼠黑質(zhì)多巴胺能神經(jīng)元數(shù)目減少24%,與對(duì)照組相比,差別有統(tǒng)計(jì)學(xué)意義(P0.05)；同時(shí)多巴胺能神經(jīng)元內(nèi)出現(xiàn)明顯的alpha-突觸核蛋白的聚集。而魚藤酮處理1月組的小鼠未觀察到明顯變化。 2.在魚藤酮處理3月組,小鼠在轉(zhuǎn)棒上的停留時(shí)間降低了35%,與對(duì)照組相比,差別有統(tǒng)計(jì)學(xué)意義(P0.05)。而魚藤酮處理1月組的小鼠未觀察到改變。 3.魚藤酮處理3月組的小鼠逃避潛伏期明顯縮短,差別有統(tǒng)計(jì)學(xué)意義(P0.05)。魚藤酮處理1月組的小鼠逃避潛伏期雖然有延長(zhǎng)趨勢(shì),但與對(duì)照組相比,差別無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。 4.在水迷宮實(shí)驗(yàn)的第5天,魚藤酮處理3月組小鼠到原平臺(tái)位置的平均距離和在相對(duì)象限的時(shí)間分別縮短16.0%和34.5%(P0.05),穿越平臺(tái)的次數(shù)和在訓(xùn)練象限的時(shí)間分別增加144%和33.4%(P0.05)。在水迷宮實(shí)驗(yàn)的第7天,到達(dá)原平臺(tái)位置的時(shí)間、到原平臺(tái)位置的平均距離和在相對(duì)象限的時(shí)間分別減少54.9%、22.2%和37.3%(P0.05),在訓(xùn)練象限的時(shí)間增加41.4%(P0.05)。這表明魚藤酮處理3月組的小鼠對(duì)平臺(tái)位置的記憶能力增強(qiáng)。 5.在水迷宮實(shí)驗(yàn)的第5天,魚藤酮處理1月組的小鼠到原平臺(tái)位置的平均距離、在相對(duì)象限的停留時(shí)間、穿越原平臺(tái)位置的次數(shù)和在訓(xùn)練象限的時(shí)間均未檢測(cè)出變化。然而,在第7天時(shí),到原平臺(tái)位置的平均距離延長(zhǎng)19.8%(P0.05),在訓(xùn)練象限中停留時(shí)間百分比降低26.4%(P0.05)。這表明魚藤酮處理1個(gè)月的小鼠記憶能力受到損害。 6.在魚藤酮處理3月組和1月組,增殖細(xì)胞核抗原和微管相關(guān)蛋白對(duì)海馬未成熟神經(jīng)元的熒光雙標(biāo)記染色呈弱陽(yáng)性,與對(duì)照組相比,差別無(wú)統(tǒng)計(jì)學(xué)意義。 本實(shí)驗(yàn)證實(shí),魚藤酮處理3月組的小鼠出現(xiàn)了PD相關(guān)的病理學(xué)和行為學(xué)改變,黑質(zhì)多巴胺能神經(jīng)元數(shù)目減少和神經(jīng)元內(nèi)alpha-突觸核蛋白的聚集,同時(shí)出現(xiàn)運(yùn)動(dòng)協(xié)調(diào)能力的障礙。本研究首次發(fā)現(xiàn)魚藤酮處理3月組的小鼠出現(xiàn)了空間學(xué)習(xí)與記憶能力的升高,而魚藤酮處理1月組的小鼠的空間記憶能力受損,這一現(xiàn)象并未伴隨海馬新生神經(jīng)元的形成,其機(jī)制尚需進(jìn)一步探討。本實(shí)驗(yàn)為研究PD中學(xué)習(xí)與記憶功能障礙提供了一定的實(shí)驗(yàn)依據(jù)和思路。
[Abstract]:Parkinson's disease (PD) is a common neurodegenerative disease. With the development of research, PD is no longer considered as a disease that occurs only in the substantia nigra-striatum system, but a disease that affects multiple nuclei of the peripheral and central nervous system. The incidence of cognitive impairment in PD patients is as high as 84%. It is.
Up to now, the etiology of PD has not been fully elucidated, although genetic and environmental factors play a role, but more than 90% of PD patients are sporadic. Epidemiological studies suggest that environmental factors are closely related to the incidence of PD, long-term exposure to pesticides in the population, the incidence of PD is higher. Rotenone is a commonly used herbicide and pesticide, can pass through the blood. Brain barrier, by inhibiting the activity of mitochondrial respiratory chain complex I, leads to the dysfunction of energy synthesis of dopaminergic neurons in substantia nigra, leading to neuronal death. Therefore, rotenone is used in the preparation of PD models. Compared with other PD animal models, rotenone-based PD models have obvious advantages, which can simulate the disease of PD. Changes in rational characteristics include a decrease in the number of dopaminergic neurons in the substantia nigra and an accumulation of alpha-synuclein in the remaining neurons.
At present, the study on the PD model of rotenone mainly focuses on the functional changes of substantia nigra-striatum system, but the changes of spatial learning and memory abilities have not been reported. The changes of dopaminergic neurons in substantia nigra and the aggregation of alpha-synuclein in neurons were observed by immunofluorescence and sulfate T staining. The changes of motor coordination in mice were observed by rotating rod method. The spatial learning and memory of mice were evaluated by Morris water maze. The changes of memory and the expression of proliferating cell nuclear antigen (PCNA) and microtubule associated protein (MICP) in hippocampal neurons were labeled by immunofluorescence double labeling to reflect the changes of immature neurons in the hippocampus.
1. The number of dopaminergic neurons in substantia nigra of mice treated with rotenone for 3 months decreased by 24% compared with the control group (P 0.05), and there was an obvious accumulation of alpha-synuclein in dopaminergic neurons.
2. In rotenone treated group for 3 months, the retention time of rotator rod in mice decreased by 35%, which was statistically significant compared with the control group (P 0.05). In rotenone treated group for 1 month, no change was observed.
3. The escape latency of mice treated with rotenone for 3 months was significantly shortened, and the difference was statistically significant (P 0.05). The escape latency of mice treated with rotenone for 1 month was prolonged, but there was no significant difference compared with the control group (P 0.05).
4. On the fifth day of the water maze experiment, the average distance from the original platform and the relative quadrant time of rotenone treated mice in the three-month group were shortened by 16.0% and 34.5% (P 0.05), the times of crossing the platform and the time of training quadrant were increased by 14.4% and 33.4% (P 0.05), respectively. The average distance of platform position and the time of relative quadrant were reduced by 54.9%, 22.2% and 37.3% (P 0.05), respectively, and the time of training quadrant was increased by 41.4% (P 0.05). This indicated that rotenone could enhance the memory of platform position in mice treated with rotenone for 3 months.
5. On the fifth day of the water maze experiment, the average distance from the original platform to the original platform of mice in rotenone treatment group for one month was not changed in the time limit of residence, the times of crossing the original platform and the time of training quadrant. However, on the seventh day, the average distance to the original platform was extended by 19.8% (P 0.05) in the training quadrant. The percentage of residence time decreased by 26.4% (P 0.05). This indicated that rotenone treatment for one month impaired the memory of mice.
6. Proliferating cell nuclear antigen (PCNA) and microtubule-associated protein (MTRP) were weakly positive for double-labeled fluorescence staining of immature hippocampal neurons in rotenone treated group for 3 months and 1 month, and there was no significant difference between the two groups.
This study demonstrated that rotenone-treated mice showed PD-related pathological and behavioral changes, decreased dopaminergic neurons in substantia nigra and aggregation of alpha-synuclein in neurons, as well as impaired motor coordination in the rotenone-treated mice. The spatial memory of mice treated with rotenone for 1 month was impaired. This phenomenon did not accompany the formation of new neurons in the hippocampus. The mechanism needs to be further explored.
【學(xué)位授予單位】：青島大學(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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本文編號(hào)：2188409