發(fā)布時間：2018-04-25 15:31

  本文選題：APP/PS1轉(zhuǎn)基因小鼠 + 3xTg-AD轉(zhuǎn)基因小鼠　； 參考：《山西醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:本研究將分別利用APPswe/PS1d E9(APP/PS1)和APPswe/PS1M146V/tau P301L(3xTg-AD)轉(zhuǎn)基因小鼠模型,應(yīng)用Western-blotting,免疫組織化學(xué)和透射電鏡的實驗手段,觀察AD模型小鼠小腦內(nèi)突觸相關(guān)蛋白表達以及超微結(jié)構(gòu)的改變,探討AD小腦突觸功能變化的可能機制,為進一步揭示小腦在AD發(fā)生中的作用提供新的理論依據(jù)。方法:選用9月齡,雄性APP/PS1雙轉(zhuǎn)基因小鼠、3xTg-AD基因小鼠與對照野生型C57BL/6J小鼠(wild type,WT)。將其分為野生型組(WT)、雙轉(zhuǎn)基因組(APP/PS1)、三轉(zhuǎn)基因組(3xTg-AD)。實驗動物由中國醫(yī)學(xué)科學(xué)院醫(yī)學(xué)實驗動物研究所基因工程平臺和The Jackson Laboratory公司提供。飼養(yǎng)環(huán)境自然晝夜節(jié)律光照,自由攝食進水,通風(fēng)良好。隨機將每組小鼠分成三個小組,分別進行免疫組化、Western-blotting、透射電鏡實驗。(1)免疫組化實驗:小鼠心臟灌注,固定后取腦組織,冷凍于-80℃,隨后切片進行免疫組織化學(xué)實驗。腦片厚度為30μm,驢血清進行封閉,一抗4℃過夜,二抗孵育,顯色,透明,封片,每步間均用PBS進行漂洗3次,每次5 min,顯微鏡下觀察比較各組小鼠小腦皮質(zhì)內(nèi)Aβ斑塊(6E10)、突觸素(Synaptophysin)、腦源性神經(jīng)營養(yǎng)因子(Brain-derived neurotrophic factor,BDNF)和其高親和力受體(Tyrosine kinase B,Trk B)的表達。(2)Western-blotting實驗:提取小鼠小腦組織蛋白,制備凝膠,蛋白加樣后,80V電壓電泳。之后轉(zhuǎn)膜,BSA封閉,一抗4℃過夜,二抗孵育,最后顯影曝光,用曝光儀分析電泳目的條帶(Synaptophysin、BDNF、Trk-B和β-actin)的表達變化。(3)透射電鏡實驗:經(jīng)心臟灌注、固定后取出小腦,將小腦皮質(zhì)切成1mm×1mm×1mm的組織塊,4%多聚甲醛固定后,用p H7.2磷酸緩沖液沖洗,置于鋨酸中后固定,雙蒸水沖洗。脫水,丙酮與包埋劑混合,室溫浸透,環(huán)氧樹脂包埋,浸透、聚合。用超薄切片機切片,醋酸鈾-檸檬酸鉛雙染,透射電鏡下觀察并拍照[1],并對突觸相關(guān)指標(biāo)進行定量分析。結(jié)果:(1)APP/PS1組和3xTg-AD組轉(zhuǎn)基因小鼠小腦皮質(zhì)內(nèi)均有少量Aβ斑塊的沉積。(2)Western-blotting實驗結(jié)果顯示,與WT組相比,APP/PS1組和3xTg-AD組轉(zhuǎn)基因小鼠小腦內(nèi)突觸素(Synaptophysin)含量明顯減少(APP/PS1:P0.01,3xTg-AD:P0.01)。免疫組化實驗結(jié)果顯示,APP/PS1組小鼠小腦分子層(P0.01)、浦肯野細胞層(P0.01)和顆粒細胞層(P0.01)中突觸素陽性表達均明顯減少;3xTg-AD組分子層(P0.01)、浦肯野細胞層(P0.01)和顆粒細胞層(P0.01)中的光密度值亦明顯減少,但與APP/PS1組相比,沒有統(tǒng)計學(xué)差異(P0.05)。兩組小鼠小腦內(nèi)浦肯野細胞形態(tài)均無明顯改變。(3)Western-blotting實驗結(jié)果顯示,與WT組相比,APP/PS1組和3xTg-AD組轉(zhuǎn)基因小鼠小腦內(nèi),BDNF的含量均明顯減少(APP/PS1:P0.01,3xTg-AD:P0.01),Trk-B含量也明顯減少(APP/PS1:P0.01,3xTg-AD:P0.01)。免疫組化實驗結(jié)果顯示,APP/PS1組和3xTg-AD組分子層浦肯野細胞層(APP/PS1:P0.01,3xTg-AD:P0.01)和顆粒細胞層內(nèi)(APP/PS1:P0.01,3xTg-AD:P0.01)陽性顆粒均明顯減少,但分子層內(nèi)BDNF陽性表達均無顯著改變(APP/PS1:P0.05,3xTg-AD:P0.05)。APP/PS1組和3xTg-AD組小腦皮質(zhì)各層內(nèi)Trk-B陽性顆粒明顯減少,相對光密度值在分子層(APP/PS1:P0.001,3xTg-AD:P0.001)、浦肯野細胞層(APP/PS1:P0.001,3xTg-AD:P0.001)以及顆粒細胞層(APP/PS1:P0.001,3xTg-AD:P0.001)內(nèi)均明顯降低。但APP/PS1組和3xTg-AD組之間沒有統(tǒng)計學(xué)差異(APP/PS1:P0.05,3xTg-AD:P0.05)。此外,在光鏡下AD小鼠浦肯野細胞有樹突變短,分枝減少等形態(tài)學(xué)變化。(4)電鏡觀察結(jié)果:APP/PS1組和3xTg-AD組轉(zhuǎn)基因小鼠均有細胞核凋亡現(xiàn)象,如電子密度增高,核固縮及異染色體邊集。兩種轉(zhuǎn)基因小鼠小腦皮質(zhì)內(nèi)均發(fā)現(xiàn)有線粒體腫脹及空泡化,甚至線粒體嵴斷裂和結(jié)構(gòu)不完整等現(xiàn)象。同時還伴有細胞骨架破壞,雙螺旋細絲樣(PHFs)等結(jié)構(gòu)變化。3xTg-AD組甚至還出現(xiàn)微管斷裂的現(xiàn)象。此外,APP/PS1組和3xTg-AD組轉(zhuǎn)基因小鼠小腦皮質(zhì)內(nèi)平行纖維-浦肯野細胞間突觸(PF-PC)的形態(tài)學(xué)參數(shù)也發(fā)生明顯變化:突觸間隙變寬(APP/PS1:P0.001,3xTg-AD:P0.001),突觸后致密區(qū)(PSD)變薄(APP/PS1:P0.001,3xTg-AD:P0.001),PSD弧度變短(P0.05)及突觸曲率變小(P0.001)。結(jié)論:(1)AD轉(zhuǎn)基因小鼠小腦內(nèi)出現(xiàn)AD特異性病理改變。(2)AD小鼠小腦內(nèi)突觸超微結(jié)構(gòu)及相關(guān)蛋白發(fā)生明顯變化,可能與BDNF合成減少有關(guān)。(3)APP/PS1組和3xTg-AD組轉(zhuǎn)基因小鼠間,小腦突觸超微結(jié)構(gòu)及相關(guān)蛋白無顯著性差異。
[Abstract]:Objective: This study will use the APPswe/PS1d E9 (APP/PS1) and APPswe/PS1M146V/tau P301L (3xTg-AD) transgenic mice model respectively, using Western-blotting, immunohistochemistry and transmission electron microscopy to observe the expression of synapse related proteins in the cerebellum and ultrastructure changes in the cerebellum of AD model mice, and explore the changes of the AD cerebellum synaptic function. The possible mechanism provides a new theoretical basis for further revealing the role of the cerebellum in the occurrence of AD. Methods: 9 month old, male APP/PS1 double transgenic mice, 3xTg-AD gene mice and control wild C57BL/6J mice (wild type, WT) were divided into wild type group (WT), double rotation genome (APP/PS1), and three transgenic genome (3xTg-AD). It was provided by the genetic engineering platform of the Institute of laboratory animals of the Chinese Academy of Medical Sciences and the The Jackson Laboratory company. The natural circadian rhythms of the feeding environment were illuminated by free feeding and well ventilated. Each group of mice was randomly divided into three groups, which were immunohistochemical, Western-blotting, transmission electron microscopy. (1) immunohistochemistry experiment: The mouse heart was perfused and frozen at -80 C, and then frozen at -80 C, then the brain slices were examined by immunohistochemistry. The thickness of the brain slices was 30 u m, the donkey serum was closed, the first anti 4 centigrade was overnight, and two was hatched, the color, the transparency, and the seal were used for 3 times and 5 min each time. The A beta spots in the cerebellar cortex of the mice were compared with the microscope. The A beta spots in the cerebellar cortex of each group were observed and compared. Under the microscope, the A beta speckles in cerebellar cortex of mice were compared. Under microscope, the A beta speckles in cerebellar cortex of mice were observed and compared. Under microscope, the A beta speckles in cerebellar cortex of mice were compared. Under microscope, the A beta speckles in cerebellar cortex of mice were compared. Under microscope, the A beta spots in cerebellar cortex of mice were compared. Mass (6E10), synaptophysin (Synaptophysin), brain derived neurotrophic factor (Brain-derived neurotrophic factor, BDNF) and its high affinity receptor (Tyrosine kinase B, Trk B). (2) Western-blotting experiment: extraction of mouse cerebellar tissue protein, preparation of gelatin, protein addition, voltage electrophoresis. Then transfer membrane, closed, one anti 4 C over Night, two anti incubation, finally exposed to exposure, using an exposure instrument to analyze the changes in the expression of the strip (Synaptophysin, BDNF, Trk-B and beta -actin). (3) transmission electron microscopy: the cerebellum was removed after the heart perfusion, the cerebellum was cut into the tissue block of 1mm * 1mm x 1mm, and 4% polyformaldehyde was fixed with P H7.2 phosphoric acid buffer solution and placed in osmium. After medium and posterior fixation, double steam rinse, dehydration, acetone and embedding agent, room temperature soaking, epoxy resin embedding, soaking, and polymerization. The ultrathin slice machine section, uranium acetate lead citrate double staining, transmission electron microscope observation and photographing [1], and quantitative analysis of synapse related indexes. Results: (1) cerebellar cortex of group APP/PS1 and 3xTg-AD group transgenic mice A small amount of A beta plaques were deposited. (2) Western-blotting experimental results showed that the content of cerebellar synaptopsin (Synaptophysin) in the APP/PS1 and 3xTg-AD groups decreased significantly (APP/PS1:P0.01,3xTg-AD:P0.01) compared with the WT group (APP/PS1:P0.01,3xTg-AD:P0.01). The immunohistochemical results showed that the cerebellar molecular layer (P0.01) and the Purkinje cell layer (P0.0) of the APP/PS1 group (P0.0). 1) the positive expression of synaptophysin in the granular cell layer (P0.01) was significantly reduced, and the light density in the 3xTg-AD group (P0.01), the Purkinje cell layer (P0.01) and the granular cell layer (P0.01) also decreased significantly, but compared with the APP/PS1 group, there was no statistical difference (P0.05). (3) the two groups of mice in the cerebellum were not significantly changed. (3) Weste The results of rn-blotting test showed that compared with the WT group, the content of BDNF in the cerebellum of APP/PS1 and 3xTg-AD mice decreased significantly (APP/PS1:P0.01,3xTg-AD:P0.01), and the content of Trk-B decreased significantly (APP/PS1:P0.01,3xTg-AD:P0.01). The results of immunohistochemical experiment showed that the molecular layer of the APP/PS1 and 3xTg-AD groups (APP/PS1:P0) (APP/PS1:P0) .01,3xTg-AD:P0.01) and granulosa cell layer (APP/PS1:P0.01,3xTg-AD:P0.01) positive particles were significantly decreased, but the positive expression of BDNF in the molecular layer was not significantly changed (APP/PS1:P0.05,3xTg-AD:P0.05) and Trk-B positive particles in each layer of the cerebellar cortex of 3xTg-AD group and.APP/PS1 group decreased significantly, and the relative light density value was in the molecular layer (APP/PS1:P0.001,3xTg). -AD:P0.001), the Purkinje cell layer (APP/PS1:P0.001,3xTg-AD:P0.001) and the granular cell layer (APP/PS1:P0.001,3xTg-AD:P0.001) were obviously decreased, but there was no statistical difference between the APP/PS1 group and the 3xTg-AD group (APP/PS1:P0.05,3xTg-AD:P0.05). In addition, the morphology of the Purkinje cells in the AD mice was shortened and the branching decreased. Changes. (4) the results of electron microscopy: nuclear apoptosis in APP/PS1 and 3xTg-AD transgenic mice, such as increased electron density, nuclear pyknosis and heterosomal edge set. Mitochondria swelling and vacuolization were found in the cerebellar cortex of the two transgenic mice, even mitochondrial crista fracture and incomplete structure. Skeleton destruction, double spiral filament like (PHFs) and other structural changes in the.3xTg-AD group even appeared microtubule fracture. In addition, the morphological parameters of the parallel fiber - Purkinje intercellular synapse (PF-PC) in the cerebellar cortex of APP/PS1 and 3xTg-AD transgenic mice also changed obviously: the synaptic gap widened (APP/PS1:P0.001,3xTg-AD:P0.001). The post touch dense region (PSD) thinning (APP/PS1:P0.001,3xTg-AD:P0.001), PSD radians shortened (P0.05) and the synaptic curvature decreased (P0.001). Conclusion: (1) there are AD specific pathological changes in the cerebellum of (1) AD transgenic mice. (2) the ultrastructure of the synapse in the cerebellum and the significant changes in the associated egg white hair in the cerebellum of AD mice may be related to the decrease of BDNF synthesis. (3) APP/PS1 and 3xT There was no significant difference in the ultrastructure of cerebellar synapses and related proteins between transgenic mice in group g-AD.

【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R749.16

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