【摘要】：目的： 褪黑素是由5-羥色胺衍生而來一種吲哚類神經(jīng)內(nèi)分泌激素，主要由哺乳動物的松果體在夜間合成和分泌。近年來研究結(jié)果表明,外源性褪黑素可參與認(rèn)知功能調(diào)節(jié)，并通過多種途徑減輕AD的病理改變。Neu-P11是一種人工合成的新型褪黑素受體激動劑，具有高褪黑素受體親和力及增強γ-氨基丁酸能神經(jīng)傳遞的特性。本研究首先檢測了Neu-P11能否增強物體識別記憶，然后基于兩種AD大鼠模型檢測了Neu-P11能否改善AD大鼠的認(rèn)知功能損傷，最后基于PC12細(xì)胞模型對Neu-P11的作用機制進行了初步探討。 方法： 1、Neu-P11對大鼠物體識別記憶的影響：大鼠物體識別測試包括兩個階段即4分鐘的訓(xùn)練期和3分鐘的檢測期。分別在上午或下午于訓(xùn)練期前2h腹腔注射溶媒、褪黑素或Neu-P11。訓(xùn)練結(jié)束后4h或24h，分別檢測大鼠的短時程與長時程記憶保持。記錄并分析各組大鼠在訓(xùn)練期的總探究時間與測試期的辨別指數(shù)。 2、Neu-P11對海馬內(nèi)微注射Aβ1-42誘導(dǎo)的AD模型大鼠認(rèn)知功能損傷的影響：海馬內(nèi)微注射Aβ1-42誘導(dǎo)產(chǎn)生AD大鼠模型，建模后第21天與30天分別進行Y迷宮與物體識別記憶測試；行為測試完成后將大鼠斷頭取腦，行組織學(xué)檢測。 3、Neu-P11對后扣帶皮層內(nèi)微注射疊氮化鈉（NaN3）誘導(dǎo)早期AD模型大鼠認(rèn)知功能損傷的影響：大鼠跳臺被動回避測試包括訓(xùn)練與檢測兩個階段。訓(xùn)練后1h將大鼠麻醉，進行后扣帶皮層內(nèi)NaN3微注射誘導(dǎo)早期AD大鼠模型。NaN3微注射后2h和6h分別進行一次Neu-P11及溶媒腹腔注射。跳臺檢測階段于訓(xùn)練結(jié)束后24h進行，檢測階段結(jié)束后2h和4h分別行自發(fā)活動及高架十字迷宮測試以評估大鼠的活動能力與焦慮水平。行為測試完成后將大鼠斷頭取腦，行組織學(xué)檢測。 4、Neu-P11對谷氨酸誘導(dǎo)PC12細(xì)胞損傷的影響：PC12細(xì)胞接種后24h，同時給予谷氨酸與Neu-P11或褪黑素處理；共孵育后24h，MTT法檢測細(xì)胞活力，流式細(xì)胞儀檢測細(xì)胞線粒體膜電位。 5、褪黑素受體拮抗劑Luzindole及5-HT1A受體拮抗劑WAY-100635對Neu-P11保護谷氨酸誘導(dǎo)PC12細(xì)胞損傷的影響：PC12細(xì)胞接種后24h，同時給予谷氨酸與Neu-P11處理；Luzindole和WAY-100635分別在Neu-P11處理的前30分鐘和前10分鐘加入到培養(yǎng)基，共孵育后24h，MTT法檢測細(xì)胞活力，，流式細(xì)胞儀檢測細(xì)胞線粒體膜電位。 結(jié)果： 1. Neu-P11對大鼠物體識別記憶的影響：在短時及長時記憶測試中，無論上午或下午給藥，Neu-P11處理組大鼠其辨別指數(shù)均顯著性高于溶媒組，而褪黑素處理組大鼠僅在下午給藥時其辨別指數(shù)顯著性高于溶媒組。在各種處理條件下，各組大鼠在訓(xùn)練階段對物體的總探究時間均無顯著性組間差異。 2. Neu-P11對海馬內(nèi)微注射Aβ1-42誘導(dǎo)的AD模型大鼠認(rèn)知功能損傷的影響：在Y迷宮測試中，各組大鼠總?cè)氡鄞螖?shù)無顯著性組間差異。AD模型組大鼠交替比率顯著性低于溶媒對照組，Neu-P11處理組大鼠交替比率顯著性高于AD模型組大鼠，而褪黑素處理組大鼠交替比率與AD模型組大鼠比較無顯著性組間差異。在物體識別測試中，各組大鼠在訓(xùn)練階段對物體的總探究時間無顯著性組間差異；在檢測階段，AD模型組大鼠辨別指數(shù)顯著性低于溶媒對照組，Neu-P11處理組大鼠辨別指數(shù)顯著性高于AD模型組大鼠，而褪黑素處理組大鼠辨別指數(shù)與AD模型組大鼠比較無顯著性組間差異。 3. Neu-P11對后扣帶皮層內(nèi)微注射NaN3誘導(dǎo)早期AD模型大鼠認(rèn)知功能損傷的影響：在跳臺被動回避測試中，各組大鼠在訓(xùn)練階段其跳臺潛伏期無顯著性組間差異；在檢測階段，溶媒對照組和Neu-P11對照組大鼠的跳臺潛伏期顯著性高于NaN3模型組大鼠；NaN3模型組大鼠其跳臺潛伏期在訓(xùn)練與檢測階段無顯著性差異；Neu-P11處理組大鼠在檢測階段的跳臺潛伏期顯著性高于訓(xùn)練階段。此外，各組大鼠的活動距離及高架十字迷宮測試指標(biāo)(開臂滯留時間百分比、開臂進入次數(shù)百分比及閉臂進入次數(shù))無顯著性組間差異。 4. Neu-P11對谷氨酸誘導(dǎo)PC12細(xì)胞損傷的影響：4mM谷氨酸可顯著性誘導(dǎo)PC12細(xì)胞的活力與線粒體膜電位水平降低；Neu-P11與褪黑素處理可顯著性改善谷氨酸誘導(dǎo)的損傷效應(yīng)。 5. Luzindole及WAY-100635對Neu-P11保護谷氨酸誘導(dǎo)PC12細(xì)胞損傷的影響：4mM谷氨酸可顯著性誘導(dǎo)PC12細(xì)胞的活力與線粒體膜電位水平降低；Neu-P11處理可顯著性改善谷氨酸誘導(dǎo)的損傷效應(yīng)。單獨Luzindole或WAY-100635處理或共處理均阻斷Neu-P11對谷氨酸誘導(dǎo)PC12細(xì)胞損傷的保護作用。 結(jié)論： Neu-P11能夠增強大鼠物體識別記憶改善AD模型大鼠認(rèn)知功能損傷，Neu-P11對AD的改善作用可能與通過激活褪黑素與5-HT1A受體進而拮抗谷氨酸誘導(dǎo)的神經(jīng)毒性相關(guān)。
[Abstract]:Purpose: Melatonin is a kind of neuroendocrine hormone derived from 5-hydroxytryptamine, which is synthesized and divided by the pineal body of the mammal at night. In recent years, the results show that the exogenous melatonin can be involved in the regulation of cognitive function, and the pathological changes of AD can be reduced by various means. Change. Neu-P11 is a synthetic novel melatonin receptor agonist, which has a high melatonin receptor affinity and a T-aminobutyric acid capable of neurotransmission. This study first examined whether Neu-P11 can enhance the recognition and memory of the object, and then, based on the two AD rat models, it is possible to improve the cognitive function damage of AD rats. Finally, the mechanism of the mechanism of Neu-P11 based on PC12 cell model is discussed. Please. Methods:1. The effect of Neu-P11 on the identification and memory of the rats: The identification and testing of the object in the rat consisted of two phases:4-minute training period and 3-minute training period. The detection period of the clock. The vehicle, melatonin or Ne were injected intraperitoneally at the morning or afternoon in the first 2 hours of the training period, respectively. U-P11. The short-term and long-term results of the rats were detected at 4 h or 24 h after the end of the training. The total study time and test period of each group of rats during the training period were recorded and analyzed. The effect of Nu-P11 on the cognitive function of rats with AD model induced by micro-injection of A-1-42 in the hippocampus: A model of AD rats induced by microinjection of A-1-42 in the hippocampus was induced by microinjection of A-1-42 in the hippocampus, and the Y-maze and the substance were respectively carried out on the 21 and 30 days after the modeling. body recognition and memory test; after the behavior test is finished, the rat is decapitated and taken into the brain The effect of NaN3 on the cognitive function of the rats with early AD model induced by NaN3 was studied by histological examination. The passive avoidance test of rats in rats was included. The training and detection were carried out in two stages. After the training, the rats were anesthetized and the NaN3 micro-injection in the cortex of the back buckle was induced. The model of early AD rats was established. Neu-P was performed at 2 h and 6 h after NaN3 microinjection. 11 and the vehicle were injected intraperitoneally. The stage of the stage detection was performed at 24 hours after the end of the training. The spontaneous activity and the elevated cross-maze test were performed at 2 and 4 hours after the end of the detection stage to evaluate the rats. Activity and anxiety levels. Rat decapitation after completion of the behavioral test The effects of 4, Neu-P11 on the injury of PC12 cells induced by glutamate were studied. The results showed that the cells were treated with glutamate and Neu-P11 or melatonin at 24 h after the inoculation of PC12 cells. The cell viability and flow cytometry were detected by MTT method after 24 h incubation. The effects of cell mitochondrial membrane potential,5, melatonin receptor antagonist Luzindole and 5-HT1A receptor antagonist WAY-100635 on the protective glutamate-induced PC12 cell injury were investigated by means of the instrument:24 h after the PC12 cell inoculation, and the glutamate and the Neu-P11 treatment at the same time; Luzindole and WAY-100635 were added to the first 30 minutes and the first 10 minutes of the Neu-P11 treatment, respectively. The cells were cultured for 24 h after incubation, and the cell viability and flow were measured by MTT method. Cytometer Detection of mitochondrial membrane potential of the cell. Results:1. The effect of 1. Neu-P11 on the identification and memory of an object in a rat: in a short and long-time memory test, the Neu-P11 treatment, whether administered in the morning or afternoon, In group rats, the discrimination index was significantly higher than that of the vehicle group, while the melatonin treatment group rats were only The differentiation index of the rats was significantly higher than that of the vehicle group at noon. Under various treatment conditions, the rats in each group were in the training stage. The effect of 2. Neu-P11 on the cognitive function of rats with AD model induced by micro-injection of A-1-42 in the hippocampus: in the Y-maze test There was no significant difference in the number of the total arms of the rats in the group. The alternating ratio of the rats in the AD model group was significantly lower than that of the vehicle control group, and the alternating ratio of the Neu-P11 treated group was significantly higher than that of the AD model group, while the alternating ratio of the melatonin treatment group to the rats There was no significant inter-group difference between the rate and the AD model group. In the object identification test, there was no significant difference in the total exploratory time of each group during the training phase; in the detection stage, the difference between the rats in the AD model group was significantly lower than that of the vehicle control group, and the Neu-P11 treatment The differentiation index of the rats in the group was significantly higher than that of the AD model group. No significant group difference between the number and the AD model group.3. The effect of neu-P11 on the cognitive function of the rats with early AD model induced by micro-injection of NaN3 in the back-buckled cortex: in the passive avoidance test of the stage, the rats in each group were in the training stage. There was no significant difference between the latency of the stage and the latency of the stage. In the detection stage, the latency of the jumping stage in the vehicle control group and the Neu-P11 control group was significantly higher than that of the NaN3 model group. The latency of the jumping stage in the NaN3 model group did not differ significantly between the training and the detection stage; the Neu-P11 treatment group was large. The incubation period of the rat in the detection stage was significantly higher than that of the training stage. In addition, the activity distance of each group and the elevated cross-maze test index (percentage of the open-arm retention time, the open-arm entry, No significant group difference was found between the number of times and the number of closed arms.4. The effect of Nu-P11 on the injury of PC12 cells induced by glutamate:4 mM glutamate significantly induced a decrease in the viability of PC12 cells and the level of mitochondrial membrane potential; Neu-P1 5. The effects of 5. Luzindole and WAY-100635 on the protective glutamate-induced PC12 cell injury were significantly improved by the treatment of melatonin.5. Luzindole and WAY-100635 had a significant effect on the protective glutamate-induced PC12 cell injury:4 mM glutamate significantly induced the decrease in the viability of PC12 cells and the level of mitochondrial membrane potential. The Neu-P11 treatment significantly improved glutamate-induced damage effects. Both the individual Luzindole or the WAY-100635 treatment or co-treatment block N eu- Conclusion: Neu-P11 can enhance the recognition and memory of the rat, improve the cognitive function of AD model, and the effect of Neu-P11 on AD may be related to the injury of PC12 cells.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R749.16

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