本文選題：側(cè)腦室注射 + 散發(fā)性阿爾茨海默病　； 參考：《南華大學(xué)》2013年碩士論文

【摘要】：目的： 鏈脲佐菌素（Streptozotocin，STZ）是一種葡萄糖胺-亞硝基脲衍生物，側(cè)腦室微注射STZ可作為研究散發(fā)性阿爾茨海默病（sporadicAlzheimer's disease，SAD）的有效動(dòng)物模型。在該模型中，STZ可引起長(zhǎng)效的大腦葡萄糖代謝和能量代謝損害，同時(shí)伴有學(xué)習(xí)記憶的進(jìn)展性損傷，如海馬依賴的空間學(xué)習(xí)記憶能力受損。然而，目前仍不清楚STZ誘導(dǎo)的SAD模型動(dòng)物是否伴有情緒相關(guān)的學(xué)習(xí)記憶能力的損傷。本研究基于大鼠條件化恐懼范式檢測(cè)了側(cè)腦室微注射STZ對(duì)大鼠情緒相關(guān)的學(xué)習(xí)記憶能力的影響，并對(duì)其可能的機(jī)制進(jìn)行了初步探討。 方法： 1.大鼠麻醉后經(jīng)雙側(cè)側(cè)腦室微量注射STZ（3mg/kg），48小時(shí)后重復(fù)注射1次；對(duì)照組大鼠微量注射同體積的檸檬酸緩沖液。第一次微注射前1天和第一次微注射后第10天檢測(cè)外周血糖濃度。 2. Morris水迷宮檢測(cè)：第一次STZ微注射后第15天進(jìn)行Morris水迷宮檢測(cè)，行為訓(xùn)練程序包括兩個(gè)階段：（1）定位航行實(shí)驗(yàn)，持續(xù)4天，每天訓(xùn)練4次，分別從4個(gè)不同的位置入水；記錄大鼠到達(dá)平臺(tái)的潛伏期。（2）空間搜索實(shí)驗(yàn)，第5天撤去平臺(tái)，大鼠從原始平臺(tái)位置的對(duì)側(cè)入水，記錄其在目標(biāo)象限停留時(shí)間。 3.場(chǎng)景性條件化恐懼反應(yīng)檢測(cè)：第一次STZ微注射后第15天進(jìn)行場(chǎng)景性條件化恐懼反應(yīng)檢測(cè)，包括習(xí)慣化、恐懼條件化、消退、重建和重建檢測(cè)等五個(gè)訓(xùn)練階段。第1天進(jìn)行習(xí)慣化訓(xùn)練，將大鼠放入A實(shí)驗(yàn)箱自由活動(dòng)10分鐘。第2天進(jìn)行恐懼條件化訓(xùn)練，大鼠在A實(shí)驗(yàn)箱中接受5次足底電刺激訓(xùn)練。第3天開始進(jìn)行恐懼消退訓(xùn)練，持續(xù)4天，將大鼠放入A實(shí)驗(yàn)箱中20分鐘，無(wú)足底電刺激。第7天給予2次足底電刺激進(jìn)行重建訓(xùn)練。第8天進(jìn)行重建檢測(cè)。條件化恐懼反應(yīng)檢測(cè)指標(biāo)為場(chǎng)景誘發(fā)的大鼠僵直時(shí)間。 4.線索性條件化恐懼反應(yīng)檢測(cè)：第一次STZ微注射后第14天進(jìn)行線索性條件化恐懼反應(yīng)檢測(cè)，包括習(xí)慣化、恐懼條件化、消退、重建和重建檢測(cè)等五個(gè)訓(xùn)練階段。第1天進(jìn)行習(xí)慣化訓(xùn)練，將大鼠分別放入A和B實(shí)驗(yàn)箱內(nèi)自由活動(dòng)10分鐘。第2天進(jìn)行恐懼條件化訓(xùn)練，大鼠在A實(shí)驗(yàn)箱中接受5次聲刺激與足底電刺激配對(duì)訓(xùn)練，聲刺激與足底電刺激同時(shí)結(jié)束。第3天開始進(jìn)行恐懼消退訓(xùn)練，持續(xù)3天，每天將大鼠放入B實(shí)驗(yàn)箱中單獨(dú)呈現(xiàn)12次聲刺激，無(wú)足底電刺激。第6天給予2次單獨(dú)足底電刺激進(jìn)行重建訓(xùn)練。第7天進(jìn)行重建檢測(cè)即將大鼠放入B實(shí)驗(yàn)箱中單獨(dú)呈現(xiàn)12次聲刺激。條件化恐懼反應(yīng)檢測(cè)指標(biāo)為聲刺激（線索）誘發(fā)的大鼠僵直時(shí)間。 5.自發(fā)活動(dòng)檢測(cè)：第一次STZ微注射后第14天進(jìn)行自發(fā)活動(dòng)檢測(cè)，將大鼠放入自發(fā)活動(dòng)檢測(cè)箱內(nèi)自由活動(dòng)30分鐘，記錄大鼠在此期間的水平活動(dòng)距離。 6.足底電擊敏感性檢測(cè)：線索性條件化恐懼反應(yīng)檢測(cè)結(jié)束后第2天進(jìn)行足底電擊敏感性檢測(cè)，將大鼠放入恐懼條件化實(shí)驗(yàn)箱內(nèi)，記錄大鼠開始出現(xiàn)注意朝向反應(yīng)、退縮反應(yīng)與驚叫反應(yīng)時(shí)的最小電流強(qiáng)度。 7.高架十字迷宮檢測(cè)：第一次STZ微注射后第14天進(jìn)行高架十字迷宮檢測(cè)，，記錄5分鐘內(nèi)大鼠進(jìn)入開放臂和閉合臂的次數(shù)以及在兩臂滯留時(shí)間。 8.生化檢測(cè)：行為學(xué)檢測(cè)完成后將大鼠處死取腦，分離海馬和杏仁核組織，采用western blot方法檢測(cè)海馬和杏仁核內(nèi)突觸素（Synaptophysin，SYP）、磷酸化環(huán)磷腺苷反應(yīng)元件結(jié)合蛋白（phosphorylated cyclicAMP response element binding protein，p-CREB）、總環(huán)磷腺苷反應(yīng)元件結(jié)合蛋白（cyclic AMP response element bindingprotein，CREB）和核轉(zhuǎn)錄蛋白κB（nuclear transcription factor kappa B，NF-κB）的表達(dá)水平。 結(jié)果： 1. STZ組大鼠外周血糖濃度與對(duì)照組比較無(wú)顯著性組間差異。 2.在Morris水迷宮檢測(cè)中，STZ組大鼠定位航行實(shí)驗(yàn)潛伏期顯著性高于對(duì)照組；在空間搜索實(shí)驗(yàn)中，STZ組大鼠在目標(biāo)象限停留時(shí)間顯著性低于對(duì)照組。 3.在場(chǎng)景性條件化恐懼反應(yīng)檢測(cè)中，STZ組大鼠在第一次消退訓(xùn)練與重建檢測(cè)階段其僵直時(shí)間百分比與對(duì)照組比較均顯著性降低；而在其余各訓(xùn)練階段，兩組大鼠僵直時(shí)間百分比均無(wú)顯著性組間差異。 4.在線索性條件化恐懼反應(yīng)檢測(cè)中，STZ組大鼠在第一次消退訓(xùn)練與重建檢測(cè)階段其僵直時(shí)間百分比與對(duì)照組比較均顯著性降低；而在其余各訓(xùn)練階段，兩組大鼠僵直時(shí)間百分比均無(wú)顯著性組間差異。 5.在自發(fā)活動(dòng)檢測(cè)中，SZT組大鼠在30分鐘內(nèi)的水平活動(dòng)距離與對(duì)照組比較無(wú)顯著性組間差異。 6.在足底電擊敏感性檢測(cè)中，SZT組大鼠注意朝向反應(yīng)、退縮反應(yīng)與驚叫反應(yīng)時(shí)的最小電流強(qiáng)度與對(duì)照組比較均無(wú)顯著性組間差異。 7.在高架十字迷宮檢測(cè)中，STZ組大鼠開放臂滯留時(shí)間百分比、進(jìn)入開放臂的次數(shù)百分比和進(jìn)入閉合臂的次數(shù)與對(duì)照組比較均無(wú)顯著性組間差異。 8. STZ組大鼠海馬和杏仁核內(nèi)SYP、p-CREB和NF-κB的表達(dá)水平均較對(duì)照組顯著性降低。 結(jié)論： 側(cè)腦室微注射STZ損害大鼠條件化恐懼記憶鞏固與重建，其機(jī)制可能與STZ誘導(dǎo)海馬和杏仁核內(nèi)SYP、p-CREB和NF-κB水平降低相關(guān)。
[Abstract]:Purpose :

In this model , it is not clear whether the induced SAD model is associated with impaired learning and memory ability . However , it is not clear whether the induced SAD model is associated with emotion - related learning and memory ability . However , it is still unclear whether the induced SAD model is associated with emotion - related learning and memory ability .

Method :

1 . After the rats were anesthetized , the rats were injected ( 3 mg / kg ) by a micro - injection at the bilateral lateral ventricle , and the injection was repeated once after 48 hours ;
The concentration of peripheral blood sugar was detected on the first day before the first micro - injection and 10 days after the first micro - injection .

2 . Morris water maze test : the Morris water maze test was performed on the 15th day after the first injection , and the behavior training program consisted of two stages : ( 1 ) positioning the navigation experiment , lasting for 4 days , training four times daily , respectively entering water from four different positions ;
The latency of the rat ' s arrival on the platform was recorded . ( 2 ) Space search experiments , the platform was removed on Day 5 , the rats were fed water from the contralateral side of the original platform position and recorded in the target quadrant residence time .

3 . Detection of Scene Conditional Fear Response : The first day , 15 days after the mini - injection , the rats were subjected to five training stages , such as habit , fear , regression , reconstruction and reconstruction . On the first day , the rats were trained and the rats were placed in A test box for 10 minutes . On day 2 , the rats were placed in A test box for 10 minutes . After the third day , the rats were put into A test box for 20 minutes without plantar electrical stimulation . The reconstruction was performed on the 8th day .

4 . Lead - induced conditioned fear response test : After the first injection , the rats were subjected to five training stages , such as habit , fear , regression , reconstruction and reconstruction . On Day 1 , the rats were placed in A and B experiment boxes for free movement for 10 minutes . After 3 days , the rats were placed in B test box for 12 times of acoustic stimulation .

5 . spontaneous activity detection : spontaneous activity detection was performed on day 14 after the first mini - injection , and the rats were placed in the spontaneous activity detection box for free activity for 30 minutes to record the horizontal movement distance during the period of the rats .

6 . Sensitivity test of foot - bottom electric shock : The sensitivity of plantar electric shock was detected on the second day after the detection of the response of the response of the clue condition , and the rats were put into the experiment box of fear condition , and the minimal current intensity in the reaction , the withdrawal reaction and the panic reaction began to appear in the recording rats .

7 . Elevated cross - maze test : A high - rack cross - maze test was performed on day 14 after the first mini - injection , and the number of rats entering the open arm and the closed arm in 5 minutes was recorded and the residence time of the two arms was recorded .

8 . Biochemical detection : After the completion of the behavioral study , the rats were sacrificed to remove the brain , separate the hippocampus and the amylic nucleus , and the expression level of Synaptophysin ( SYP ) , phosphorylated cyclic AMP response element binding protein ( p - CREB ) , cyclic AMP response element binding protein ( CREB ) and nuclear transcription factor kappa B ( NF - 魏B ) were detected by western blot .

Results :

1 . There was no significant difference between the peripheral blood sugar concentration and the control group .

2 . In Morris water maze test , the latent period of the rats ' positioning navigation was significantly higher than that in the control group .
In the space search experiment , the retention time of the rats in the target quadrant was significantly lower than that in the control group .

3 . The percentage of stiffness and the percentage of stiffness in the control group were significantly lower than those in the control group during the first time of regression training and reconstruction .
There was no significant difference between the two groups during the training period .

4 . Compared with the control group , the percentage of stiffness and the percentage of the control group were significantly lower than those in the control group .
There was no significant difference between the two groups during the training period .

5 . In spontaneous activity test , there was no significant difference in the level of activity between SZT group and control group within 30 minutes .

6 . In the detection of electroshock sensitivity of foot bottom , the rats of SZT group showed no significant difference between the minimum current intensity and the control group when the response was observed .

7 . There was no significant difference between the percentage of open arms and the number of open arms and the number of open arms and the number of closed arms compared with the control group .

8 . The expression levels of SYP , p - CREB and NF - 魏B were significantly lower in the hippocampus and almond nuclei than in the control group .

Conclusion :

The mechanism may be related to the decrease of SYP , p - CREB and NF - 魏B levels in the hippocampus and in the apricot kernel .

【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R749.16

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