發(fā)布時間：2018-03-18 08:33

  本文選題：阿爾茨海默病　切入點：星形膠質(zhì)細胞　出處：《寧波大學》2013年碩士論文　論文類型：學位論文

【摘要】：目的：通過星形膠質(zhì)細胞培養(yǎng)和激光共聚焦成像的方法，研究阿爾茨海默病(Alzheimer’s disease, AD)的模型鼠——APPswe/PS1ΔE9轉(zhuǎn)基因鼠以及老年星形膠質(zhì)細胞溶酶體對可溶性β-淀粉樣蛋白(β-Amyloid protein42，Aβ42)的降解能力是否下降。探討細胞內(nèi)PH改變是否影響星形膠質(zhì)細胞溶酶體對外源性Aβ42的降解能力。通過星型膠質(zhì)細胞溶酶體對Aβ42降解機制的研究，進一步了解AD可能的發(fā)病機制，為尋找AD的治療靶點提供理論和實驗依據(jù)。 方法：通過PCR的方法鑒定APPswe/PS1ΔE9雙轉(zhuǎn)基因鼠，，取新生第一天的小鼠進行原代星形膠質(zhì)細胞的培養(yǎng)。用帶綠色熒光的Aβ42(Hilyte Fluor488-labled Amyloid-β protein42，Aβ42)處理體外培養(yǎng)第7天(day7in vitro, DIV7)，體外培養(yǎng)第21天(day21in vitro,DIV21)的星形膠質(zhì)細胞，孵育30min。用l μM的溶酶體的染料Lysotracker DND Red99(DND-99)對溶酶體染色，激光共聚焦顯微鏡觀察，分析APP/PS1雙轉(zhuǎn)基因鼠星形膠質(zhì)細胞溶酶體與Aβ42共定位率和正常組比是否有差別。同時分析DIV7星形膠質(zhì)細胞內(nèi)Aβ42和溶酶體的熒光強度隨時間變化情況；觀察不同組星形膠質(zhì)細胞內(nèi)吞Aβ42的動態(tài)變化過程。用NH4Cl處理改變細胞內(nèi)PH后，分析AD組星形膠質(zhì)細胞溶酶體與外源性Aβ42共定位率是否有差異。 結果： 1.星形膠質(zhì)細胞內(nèi)吞外源性Aβ42后30min，定位于溶酶體。 2.經(jīng)Aβ42和DND-99孵育30min后，Aβ42和溶酶體的熒光強度在DIV7星形膠質(zhì)細胞AD組和正常組間均沒有差異，說明AD組和正常組DIV7星形膠質(zhì)細胞對Aβ42的內(nèi)吞沒有差異。隨著時間變化，AD組DIV7星形膠質(zhì)細胞溶酶體與外源性Aβ42的共定位率與正常組比均顯著偏高，37℃孵育2h后， AD組DIV7星形膠質(zhì)細胞內(nèi)Aβ42熒光強度高于正常組，AD組DIV7星形膠質(zhì)細胞內(nèi)溶酶體熒光強度低于正常組。 3. AD組DIV21星形膠質(zhì)細胞溶酶體和Aβ42的熒光強度與正常組DIV21星形膠質(zhì)細胞比均下降，正常組DIV21星形膠質(zhì)細胞溶酶體和Aβ42的熒光強度與正常組DIV7比均下降。 4.經(jīng)25mM的NH4Cl溶液處理后，AD組星形膠質(zhì)細胞溶酶體與Aβ42的共定位率下降。 結論： 1. DIV7星形膠質(zhì)細胞內(nèi)吞外源性Aβ42后30min定位于溶酶體。 2. AD小鼠DIV7星形膠質(zhì)細胞溶酶體對外源性Aβ42的降解能力下降。 3. DIV21老膠質(zhì)細胞對外源性Aβ42的內(nèi)吞能力下降，DIV21老星形膠質(zhì)細胞溶酶體活性下降。 4. NH4Cl能加強星形膠質(zhì)細胞溶酶體對外源性Aβ42的降解。
[Abstract]:Objective: to study the methods of astrocyte culture and laser confocal imaging. To study the degradation of soluble 尾 -amyloid protein (尾 -Amyloid protein 42 A 尾 42) by APPsweP PS1 螖 E9 transgenic mice and senile astrocyte lysosomes in Alzheimer's disease (ADD) model mice. To investigate whether the changes of intracellular PH affect the degradation of soluble 尾 -amyloid protein A 尾 42.To investigate the effects of intracellular PH changes on the degradation of soluble 尾 -amyloid protein (尾 -Amyloid protein 42A 尾 42) in mice with Alzheimer's disease (ADD). The degradation of exogenous A 尾 42 by lysosomes in astrocytes, and the mechanism of degradation of A 尾 42 by astrocyte lysosomes. To further understand the possible pathogenesis of AD and to provide theoretical and experimental evidence for finding therapeutic targets for AD. Methods: APPswe/PS1 螖 E9 transgenic mice were identified by PCR. Primary astrocytes were cultured in mice on the first day of birth. Astrocytes were cultured in vitro with A 尾 42 Fluor488-labled Fluor488-labled A myloid- 尾 42A 尾 42) on the 7th day of culture, and on the 21st day of culture with day21in Vitro-DIV21), and the astrocytes were cultured on the 21st day in vitro, and the astrocytes were cultured on day 7 and day 21 respectively, and the astrocytes were cultured in vitro with A 尾 42n Hilyte Fluor488-labled Amyloid- 尾 42 (A 尾 42). The astrocytes were cultured in vitro on the 7th day. After incubation for 30 min, the lysosomes were stained with 1 渭 M lysosomal dye Lysotracker DND Red99 DND-99, and observed by confocal laser microscopy. To analyze whether the co-localization rate of lysosome and A 尾 42 of APP/PS1 double transgenic mice is different from that of normal group, and to analyze the change of fluorescence intensity of A 尾 42 and lysosome in DIV7 astrocytes with time. To observe the dynamic changes of endocytosis A 尾 42 in astrocytes of different groups. After treating with NH4Cl to change the intracellular PH, we analyzed whether there were differences in the co-localization rate of lysosomes between astrocytes and exogenous A 尾 42 in AD group. Results:. 1. After endocytosis of A 尾 42, astrocytes were located in lysosomes for 30 min. 2. After incubation with A 尾 42 and DND-99 for 30 minutes, the fluorescence intensity of A 尾 42 and lysosome was not different between AD group and normal group of DIV7 astrocytes. The results showed that the uptake of A 尾 42 by DIV7 astrocytes in AD group and normal group was different, and the co-localization rate of lysosome of DIV7 astrocytes and exogenous A 尾 42 in AD group was significantly higher than that in normal group after incubation at 37 鈩

本文編號：1628820