發(fā)布時(shí)間：2018-03-07 11:22

  本文選題：血管性癡呆　切入點(diǎn)：小鼠　出處：《河北醫(yī)科大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:血管性癡呆(vascular dementia,VD)主要是因腦組織血液循環(huán)障礙所致的一種獲得性、進(jìn)行性認(rèn)知功能障礙綜合征,是最常見(jiàn)的老年期癡呆原因之一。隨著社會(huì)人口的老齡化和腦血管疾病發(fā)病率的增高,VD的發(fā)病率也隨之增高,嚴(yán)重地威脅著老年人的身體健康和生存質(zhì)量。 但是,VD確切的發(fā)病機(jī)制尚不清楚,臨床也缺乏有效的預(yù)防和治療藥物。建立理想的VD動(dòng)物模型是探討其發(fā)病機(jī)制,研制有效的防治藥物并進(jìn)行評(píng)價(jià)的關(guān)鍵。目前常用的VD動(dòng)物模型有:血管阻斷VD模型、血管內(nèi)栓塞VD模型、高脂血癥VD模型、VD自發(fā)模型和光化學(xué)誘導(dǎo)VD模型等,均以模擬人類VD發(fā)病特點(diǎn)為基礎(chǔ)而建立,種類較多,但效果各異,尚缺乏穩(wěn)定可靠的VD動(dòng)物模型。 用于VD研究的動(dòng)物首先應(yīng)有與人類相似的腦血管解剖特點(diǎn),其次應(yīng)該便于進(jìn)行學(xué)習(xí)記憶的行為學(xué)測(cè)試。考慮到小鼠在具備上述要求的同時(shí),價(jià)格低廉,繁殖快,生存率高,實(shí)驗(yàn)操作簡(jiǎn)便。因此,本實(shí)驗(yàn)選用昆明小鼠,建立五種VD小鼠模型,并通過(guò)避暗箱實(shí)驗(yàn)和Morris水迷宮對(duì)上述五種模型中小鼠學(xué)習(xí)記憶的改變進(jìn)行檢測(cè),同時(shí)應(yīng)用硫堇染色觀察小鼠海馬神經(jīng)元損傷情況,再結(jié)合小鼠大腦的大體改變對(duì)上述各VD模型的效果進(jìn)行比較,以篩選出較為穩(wěn)定可靠的VD模型,為后續(xù)VD發(fā)病機(jī)制及有效防治措施的研究奠定基礎(chǔ)。 方法:將105只28-32克健康雄性昆明小鼠隨機(jī)分為以下7組(n=15): (1)正常組:正常小鼠,不做任何處理。 (2)Sham組:只暴露雙側(cè)頸總動(dòng)脈,但不阻斷血流。 (3)10分鐘模型組:分別暴露雙側(cè)頸總動(dòng)脈,反復(fù)夾閉雙側(cè)頸總動(dòng)脈3次,每次夾閉10分鐘,每次間隔10分鐘,于第一次夾閉頸總動(dòng)脈的同時(shí)尾動(dòng)脈放血0.3 ml。 (4)20分鐘模型組:分別暴露雙側(cè)頸總動(dòng)脈,反復(fù)夾閉雙側(cè)頸總動(dòng)脈3次,每次夾閉20分鐘,每次間隔10分鐘,于第一次夾閉頸總動(dòng)脈的同時(shí)尾動(dòng)脈放血0.3 ml。 (5)30分鐘模型組:分別暴露雙側(cè)頸總動(dòng)脈,反復(fù)夾閉雙側(cè)頸總動(dòng)脈3次,每次夾閉30分鐘,每次間隔10分鐘,于第一次夾閉頸總動(dòng)脈的同時(shí)尾動(dòng)脈放血0.3 ml。 (6)左側(cè)結(jié)扎模型組:分別暴露雙側(cè)頸總動(dòng)脈,左側(cè)頸總動(dòng)脈永久性結(jié)扎,右側(cè)頸總動(dòng)脈反復(fù)夾閉3次,每次夾閉30分鐘,每次間隔10分鐘。 (7)右側(cè)結(jié)扎模型組:分別暴露雙側(cè)頸總動(dòng)脈,右側(cè)頸總動(dòng)脈永久性結(jié)扎,左側(cè)頸總動(dòng)脈反復(fù)夾閉3次,每次夾閉30分鐘,每次間隔10分鐘。 各組動(dòng)物在相同環(huán)境下飼養(yǎng)4周。其中正常組動(dòng)物直接于飼養(yǎng)4周后,其余各組動(dòng)物于術(shù)后4周時(shí),首先通過(guò)避暗箱實(shí)驗(yàn)和Morris水迷宮檢測(cè)其學(xué)習(xí)記憶能力的改變;待行為學(xué)實(shí)驗(yàn)結(jié)束后取材,采集小鼠大腦大體標(biāo)本圖像,觀察其大體改變,同時(shí)應(yīng)用硫堇染色,觀察海馬神經(jīng)元損傷情況。 結(jié)果: 1小鼠學(xué)習(xí)記憶能力的改變 1.1被動(dòng)逃避反應(yīng)的改變——避暗箱實(shí)驗(yàn) 飼養(yǎng)或術(shù)后第29天,開(kāi)始對(duì)所有動(dòng)物進(jìn)行避暗箱測(cè)試,采用被電次數(shù)(次/3 min)和避暗潛伏期(s)作為學(xué)習(xí)記憶的檢測(cè)指標(biāo)。 結(jié)果顯示:正常組小鼠的被電次數(shù)是1.7±1.0。與正常組相比,Sham組小鼠的被電次數(shù)(1.8±0.8)無(wú)顯著變化。與正常組和Sham組相比,各模型組的被電次數(shù)均有增加的趨勢(shì),但無(wú)統(tǒng)計(jì)學(xué)意義。 正常組小鼠的避暗潛伏期為267.0±52.9。與正常組相比,Sham組小鼠的避暗潛伏期(247.9±101.5)無(wú)顯著性變化,而左側(cè)結(jié)扎模型組小鼠的避暗潛伏期(198.9±103.6)和右側(cè)結(jié)扎模型組小鼠的避暗潛伏期(186.4±111.5)均顯著縮短(P0.05)。其余10分鐘模型組、20分鐘模型組和30分鐘模型組,與正常組和Sham組相比,均有一定的縮短趨勢(shì),但無(wú)統(tǒng)計(jì)學(xué)意義。 以上結(jié)果表明,左側(cè)結(jié)扎模型組小鼠和右側(cè)結(jié)扎模型組小鼠在被動(dòng)逃避反應(yīng)中顯示出明顯的學(xué)習(xí)記憶能力的下降。 1.2空間學(xué)習(xí)記憶能力的改變——Morris水迷宮實(shí)驗(yàn) 避暗箱實(shí)驗(yàn)結(jié)束后,對(duì)所有實(shí)驗(yàn)動(dòng)物進(jìn)行Morris水迷宮測(cè)試,以定向航行階段的尋臺(tái)潛伏期(s)和空間搜索階段的平臺(tái)所在象限滯留時(shí)間(s)、尋臺(tái)次數(shù)(次)作為學(xué)習(xí)記憶的檢測(cè)指標(biāo)。 結(jié)果顯示:在定向航行實(shí)驗(yàn)階段,正常組小鼠定向航行5 d的尋臺(tái)潛伏期逐漸縮短。與正常組小鼠相比,Sham組小鼠的尋臺(tái)潛伏期無(wú)顯著性變化。與正常組、Sham組、10分鐘模型組和20分鐘模型組相比,30分鐘模型組和右側(cè)結(jié)扎模型組小鼠的尋臺(tái)潛伏期均顯著延長(zhǎng)(P0.05);并且,右側(cè)結(jié)扎模型組小鼠的尋臺(tái)潛伏期還顯著長(zhǎng)于左側(cè)結(jié)扎模型組小鼠的尋臺(tái)潛伏期(P0.05);10分鐘模型組、20分鐘模型組和左側(cè)結(jié)扎模型組小鼠的尋臺(tái)潛伏期均無(wú)顯著性變化。 在空間搜索實(shí)驗(yàn)階段,60 s內(nèi)正常組小鼠的平臺(tái)所在象限滯留時(shí)間是21.3±8.1,尋臺(tái)次數(shù)是4.1±2.1。與正常組相比,Sham組小鼠的平臺(tái)象限滯留時(shí)間(18.1±8.8)、尋臺(tái)次數(shù)(2.7±1.8),均無(wú)顯著性差異;而10分鐘模型組的平臺(tái)象限滯留時(shí)間(15.1±7.9)、尋臺(tái)次數(shù)(2.5±1.9),30分鐘模型組的平臺(tái)象限滯留時(shí)間(14.4±6.0)、尋臺(tái)次數(shù)(2.1±1.9),左側(cè)結(jié)扎模型組的平臺(tái)象限滯留時(shí)間(15.1±7.3)、尋臺(tái)次數(shù)(2.0±1.6),以及右側(cè)結(jié)扎模型組的平臺(tái)象限滯留時(shí)間(14.0±5.8)、尋臺(tái)次數(shù)(2.2±1.4)均顯著減少(P0.05)。與Sham組相比,僅右側(cè)結(jié)扎模型組的平臺(tái)象限滯留時(shí)間顯著縮短(P0.05)。20分鐘模型組的平臺(tái)象限滯留時(shí)間(16.9±8.8)、尋臺(tái)次數(shù)(3.1±2.1)與正常組和Sham組相比,均無(wú)顯著性變化。 以上結(jié)果表明,10分鐘模型組、30分鐘模型組、左側(cè)結(jié)扎模型組和右側(cè)結(jié)扎模型組小鼠的空間學(xué)習(xí)記憶能力均有不同程度的下降,其中以右側(cè)結(jié)扎模型組小鼠下降最為顯著。 2小鼠大腦的形態(tài)學(xué)改變 各組小鼠于行為學(xué)實(shí)驗(yàn)后取材,首先在體視顯微鏡下觀察小鼠大腦的大體外觀改變。結(jié)果顯示:正常組小鼠的大腦外觀晶瑩剔透,腦組織飽滿、無(wú)缺損。Sham組小鼠的大腦外觀與正常組小鼠相似,腦組織飽滿、無(wú)明顯缺損。各模型組中,除了10分鐘模型組小鼠未見(jiàn)明顯的腦組織缺損外,其余4個(gè)模型組中,均有部分小鼠的腦組織出現(xiàn)明顯的梗死灶。其中,20分鐘模型組中出現(xiàn)明顯梗死灶的小鼠占20%;30分鐘模型組中占13%;左側(cè)結(jié)扎模型組中占13%;右側(cè)結(jié)扎模型組中占33%。 以上結(jié)果表明,右側(cè)結(jié)扎模型組小鼠腦組織大體損傷的成功率最高。 3小鼠海馬的組織病理學(xué)改變 對(duì)整個(gè)海馬的組織病理學(xué)評(píng)價(jià)結(jié)果顯示:正常組和Sham組小鼠海馬各區(qū)錐體神經(jīng)元排列致密、整齊,胞核飽滿,核仁清晰;組織學(xué)分級(jí)(Histological grade,HG)均為0～Ⅱ級(jí),其中HG 0～Ⅰ級(jí)的分別占87%和93%。 相比之下,10分鐘模型組和20分鐘模型組小鼠海馬各區(qū)錐體細(xì)胞排列較整齊,核固縮現(xiàn)象較少;其中,10分鐘模型組小鼠HG 0～Ⅰ級(jí)的占60%,HGⅡ級(jí)的占33%,HGⅢ級(jí)的占7%;20分鐘模型組小鼠HG 0～Ⅰ級(jí)的占66%,HGⅡ級(jí)的占27%,HGⅢ級(jí)的占7%;與正常組和Sham組相比,此兩組的HG均有升高的趨勢(shì),但無(wú)統(tǒng)計(jì)學(xué)意義。 30分鐘模型組部分小鼠海馬可見(jiàn)片段性的錐體細(xì)胞缺失,HG 0～Ⅰ級(jí)的占67%,HGⅡ級(jí)的占20%,HGⅢ級(jí)的占13%;與正常組和Sham組相比,該組的HG也僅有升高的趨勢(shì),但無(wú)統(tǒng)計(jì)學(xué)意義。 左側(cè)結(jié)扎模型組和右側(cè)結(jié)扎模型組小鼠海馬片段性的錐體細(xì)胞缺失顯著增多;其中,左側(cè)結(jié)扎模型組HG 0～Ⅰ級(jí)的占47%,HGⅡ級(jí)的占33%,HGⅢ級(jí)的占20%;右側(cè)結(jié)扎模型組HG 0～Ⅰ級(jí)的占47%,HGⅡ級(jí)的占26%,HGⅢ級(jí)的占27%;與正常組和Sham組相比,此兩組的HG均顯著升高(P0.05),并且右側(cè)結(jié)扎模型組小鼠的HG還顯著高于30分鐘模型組小鼠的HG(P0.05)。 以上結(jié)果表明,左側(cè)結(jié)扎模型組和右側(cè)結(jié)扎模型組造成的小鼠海馬神經(jīng)元損傷較為嚴(yán)重,并且,以右側(cè)結(jié)扎模型組海馬神經(jīng)元損傷最為嚴(yán)重。 結(jié)論: 本實(shí)驗(yàn)建立的5種VD小鼠模型中,右側(cè)結(jié)扎模型組造成的小鼠大腦大體損傷的成功率最高,引起的海馬神經(jīng)元的損傷最為嚴(yán)重;該組小鼠的被動(dòng)逃避反應(yīng)和空間學(xué)習(xí)記憶能力的降低最為顯著,且表現(xiàn)較為穩(wěn)定。
[Abstract]:Objective: vascular dementia (vascular dementia VD) is mainly due to an acquired brain blood circulation disorder caused by the progressive cognitive dysfunction syndrome, is one of the most common cause of dementia in the elderly. With the aging of the population and the incidence of cerebrovascular diseases increased, the incidence of VD is also increased, a serious threat to the health and quality of life of the elderly.
However, the exact mechanism of VD is not clear, and lack of clinical prevention and treatment of drug effective. Establish the ideal animal model of VD is to explore its pathogenesis, key to the development of effective drugs and evaluation. At present VD animal models are commonly used: VD vascular occlusion model, endovascular embolization in VD model. Hyperlipidemia, VD model, VD model and VD model of photochemically induced spontaneous, both to simulate the characteristics of human VD pathogenesis based, more categories, but the effect is different, the lack of VD animal model is stable and reliable.
For the characteristics of cerebral vascular anatomy research of VD animal should first be similar to humans, secondly should facilitate the learning and memory behavior were tested. Considering the mice in with the above requirements at the same time, low price, fast breeding, high survival rate, the operation is simple. Therefore, the selection of Kunming mice, five VD mice were established model, and by avoiding dark box test and Morris water maze to change the above five models of learning and memory of mice were detected at the same time were observed in mice hippocampal neurons injury by thionine, combined with the change of the mouse brain in each VD model to compare the effects of selected VD model is more stable and reliable. The study laid the foundation for the follow-up of VD pathogenesis and effective prevention and control measures.
Methods: 105 28-32 gram healthy male Kunming mice were randomly divided into 7 groups (n=15).
(1) normal group: normal mice, do not do any treatment.
(2) group Sham: only bilateral common carotid artery was exposed, but the blood flow was not blocked.
(3) 10 minutes model group: exposed bilateral common carotid arteries respectively, 3 times of bilateral common carotid arteries were clipped repeatedly, 10 minutes per clip and 10 minutes intervals, the first time to clamp the common carotid artery, the tail artery was bleeding 0.3 ml..
(4) 20 minutes model group: exposed bilateral common carotid arteries respectively, 3 times of bilateral common carotid arteries were clipped repeatedly, 20 minutes per clip and 10 minutes intervals, the first time to clamp the common carotid artery, the tail artery was bleeding 0.3 ml..
(5) 30 minutes model group: exposed bilateral common carotid arteries respectively, 3 times of bilateral common carotid arteries were clipped repeatedly, 30 minutes per clip and 10 minutes intervals, the first time to clamp the common carotid artery, the tail artery was bleeding 0.3 ml..
(6) the left ligation model group was exposed to bilateral common carotid arteries, the left common carotid artery was ligated permanently, and the right common carotid artery was clipped repeatedly for 3 times. Each clamp was closed for 30 minutes, and the interval between each time was 10 minutes.
(7) the right side ligation model group: the bilateral common carotid arteries were exposed respectively, the right common carotid artery was permanently ligated, and the left common carotid artery was clipped repeatedly for 3 times. Each clamp was closed for 30 minutes, and the interval between each time was 10 minutes.
Each animal feeding for 4 weeks in the same environment. The normal animal directly after 4 weeks feeding, the rest in each animal after 4 weeks, first by avoiding dark box test and Morris water maze test the ability of learning and memory behavior change; to the end of the experiment were collected, the mouse brain specimen images. To observe the general change, at the same time using thionin staining, observed the injury of hippocampal neurons.
Result:
1 changes in learning and memory ability of mice
1.1 change of passive avoidance response: dark box experiment
On the twenty-ninth day after raising or after operation, all animals were tested in the dark box test. The number of times of electricity (/3 min) and dark latency (s) were used as indicators for learning and memory.
The results showed that the number of times of electric charge in normal group was 1.7 + 1.0.. Compared with the normal group, the number of times of electric shock in group Sham (1.8 + 0.8) did not change significantly. Compared with the normal group and Sham group, the number of times of electric charge increased in all models, but there was no statistical significance.
In the normal group the dark incubation period was 267 + 52.9. compared with the normal group, Sham group of mice the avoiding latency was (247.9 + 101.5) no significant changes, but the left ligation model mice stepthrough latency (198.9 + 103.6) and right ligation model mice stepthrough latency (186.4 + 111.5) were significantly shortened (P0.05). The remaining 10 minutes group, 20 minutes and 30 minutes in model group compared with model group, normal group and Sham group had a tendency to reduce, but not statistically significant.
The above results showed that the mice in the left ligation model group and the right ligation model group showed a significant decline in the learning and memory ability in the passive avoidance response.
The change of learning and memory in 1.2 space -- Morris water maze experiment
After the end of the dark box experiment, the Morris water maze test was carried out on all the experimental animals. The incubation period (s) of the navigation phase and the quadrant residence time (s) of the platform in the spatial search stage were selected. The number of search times (Times) was used as a detection index for learning and memory.
The results showed that: in the navigation experiment stage, normal mice navigation 5 d eacape latency shortened gradually. Compared with the normal mice, Sham mice eacape latency had no significant change. With the normal group, Sham group, model group, 10 minutes and 20 minutes compared to the model group, the model group and 30 minutes the ligation model mice eacape latency were significantly prolonged (P0.05); and, on the right side of ligation model mice eacape latency significantly longer than the left ligation model mice eacape latency (P0.05); 10 minutes 20 minutes in model group, model group and ligation of left model group mice were eacape latency no significant change.
鍦ㄧ┖闂存悳绱㈠疄楠岄樁孌，

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