【摘要】：目的：阿爾茨海默病(Alzheimer's Disease, AD)是一種最常見的神經(jīng)退行性疾病,主要臨床表現(xiàn)為認(rèn)知和記憶功能進(jìn)行性減退。細(xì)胞骨架蛋白神經(jīng)絲(neurofilaments, NFs)是神經(jīng)元特異的中間絲成分,由三種不同分子量的亞單位組裝成的多聚體。異常過(guò)度磷酸化的神經(jīng)絲聚集在細(xì)胞軸突,是AD腦中神經(jīng)元纖維纏結(jié)(NFT)的一種重要成分和早期的病理改變,但其發(fā)病機(jī)制目前尚不清楚。除了磷酸化,NFs蛋白還受細(xì)胞內(nèi)葡萄糖代謝產(chǎn)生的O-糖基化(O-GlcNAcylation)修飾,我們的最新研究發(fā)現(xiàn)：在細(xì)胞水平NFs的糖基化和磷酸化之間存在相互調(diào)節(jié),AD病人的腦神經(jīng)細(xì)胞內(nèi)聚集的異常磷酸化的NFs其O-糖基化明顯減少。本課題是在上述研究結(jié)果的基礎(chǔ)上,主要研究神經(jīng)絲的O-糖基化失調(diào)對(duì)其磷酸化和組裝的影響,探討神經(jīng)絲過(guò)度磷酸化的發(fā)病機(jī)制。 方法：我們主要采用體內(nèi)和體外實(shí)驗(yàn)觀察神經(jīng)絲的O-糖基化的改變對(duì)其磷酸化和組裝的影響。4-6月齡清潔級(jí)雄性SD大鼠35只大鼠平均分5組,每組7只,腦室內(nèi)分別注射Alloxan (OGT抑制劑,2mg/kg)、選擇性葡萄糖胺酶抑制劑NAG-AE (NAG-thiazoline,5mg/kg)組、Don (HBP途徑抑制劑,2mg/kg)、 OA (PP2A抑制劑,50ng/kg)、實(shí)驗(yàn)對(duì)照組(生理鹽水),空白對(duì)照鼠3只。24小時(shí)后處死大鼠取腦,采用蛋白免疫印跡檢測(cè)腦組織NFs蛋白的糖基化和磷酸化水平；采用腦勻漿液高速離心和神經(jīng)絲體外組裝技術(shù),結(jié)合透射電鏡和免疫印跡方法,定性和定量對(duì)比不同糖基化水平的鼠腦NFs的體外組裝的情況和形態(tài)。進(jìn)一步我們采用體外酶化學(xué)技術(shù),用β-乙酰糖胺酶去除腦組織勻漿液蛋白質(zhì)的糖基化,用電鏡技術(shù)觀察神經(jīng)絲組裝的結(jié)構(gòu),探討是NFs糖基化本身還是通過(guò)繼發(fā)的磷酸化影響鼠腦NFs組裝。 結(jié)果：我們的預(yù)試驗(yàn)表明神經(jīng)絲提取效率高,純度較高,電鏡觀察組裝后神經(jīng)絲由直徑10nm左右的纖維主干以及由主干周期性發(fā)出的側(cè)臂組成,側(cè)臂一般較短,大小及結(jié)構(gòu)與其它方法提取的神經(jīng)絲相同。在體實(shí)驗(yàn)中,與對(duì)照組相比Alloxan、DON、OA組神經(jīng)絲蛋白O-糖基化水平降低而磷酸化增加,神經(jīng)絲較短且組裝的比例明顯減低；而NAG-Ae處理與對(duì)照組相比,O-糖基化水平明顯增加而磷酸化水平是明顯降低,神經(jīng)絲纖維呈長(zhǎng)絲狀,組裝的比例較高。提示神經(jīng)絲的O-糖基化的改變對(duì)神經(jīng)絲的組裝有一定的影響。進(jìn)一步在體外實(shí)驗(yàn),我們只改變NFs的糖基化但不改變其磷酸化水平,電鏡負(fù)染神經(jīng)絲與對(duì)照組相比,神經(jīng)絲的看起來(lái)較粗糙,仍然組裝成較長(zhǎng)的絲,提示單純?nèi)ヌ腔瘜?duì)組裝影響不明顯。 結(jié)論：體內(nèi)O-糖基化的改變影響NFs的磷酸化,減少的O-糖基化可能通過(guò)影響其磷酸化和神經(jīng)絲的組裝而影響其結(jié)構(gòu)和功能,導(dǎo)致AD神經(jīng)細(xì)胞的退行性變的發(fā)生。該研究結(jié)果為AD發(fā)病機(jī)制理論研究和藥物的研發(fā)提供新的思路。
[Abstract]:Objective: Alzheimer's disease (Alzheimer's Disease, AD) is one of the most common neurodegenerative diseases. The cytoskeletal protein neurofilament (neurofilaments, NFs) is a neuron-specific intermediate filament composed of three different molecular weight subunits. Abnormal hyperphosphorylation of neurofilament is an important component and early pathological change of neurofibrillary tangles (NFT) in AD brain, but its pathogenesis is still unclear. In addition to phosphorylation, NFs proteins are also modified by Oglycosylation (O-GlcNAcylation) produced by glucose metabolism in cells. Our latest study has found that there is a mutual regulation between glycosylation and phosphorylation of NFs at the cellular level. Abnormal phosphorylation of NFs in brain nerve cells of AD patients decreased significantly in O-glycosylation. On the basis of the above research results, this paper mainly studies the effect of the disorder of O-glycosylation on the phosphorylation and assembly of neurofilament, and probes into the pathogenesis of neurofilament hyperphosphorylation. Methods: the effects of changes of O-glycosylation of neurofilament on phosphorylation and assembly of neurofilament were observed in vivo and in vitro. 35 male SD rats aged 4-6 months were divided into 5 groups with 7 rats in each group. Intraventricular injection of Alloxan (OGT inhibitor (2mg/kg), selective glucosaminase inhibitor NAG-AE (NAG-thiazoline,5mg/kg), Don (HBP pathway inhibitor, 2mg/kg), OA (PP2A inhibitor, 50ng/kg, respectively. The control group (normal saline) and the blank control group (3 rats) were killed 24 hours later. The glycosylation and phosphorylation of NFs protein in brain tissue were detected by Western blot. High speed centrifugation of brain homogenate, in vitro assembly of neurofilament, transmission electron microscopy and Western blotting were used to qualitatively and quantitatively compare the in vitro assembly and morphology of brain NFs with different glycosylation levels. Furthermore, we used in vitro enzyme chemistry to remove the glycosylation of protein from brain homogenate by 尾 -acetylglycosaminase, and observed the structure of neurofilament assembly by electron microscopy. To investigate whether NFs glycosylation itself or secondary phosphorylation affects brain NFs assembly. Results: our pre-test showed that the extraction efficiency of neurofilament was high and the purity was high. Electron microscopic observation showed that the nerve filament was composed of the fiber trunk about diameter 10nm and the lateral arm which was issued periodically by the trunk, and the lateral arm was generally short. The size and structure were the same as those obtained by other methods. In vivo, compared with the control group, the level of O-glycosylation of neurofilament protein in Alloxan,DON,OA group was decreased, the phosphorylation was increased, and the proportion of neurofilament was shorter and the proportion of assembly was significantly lower than that in control group. Compared with the control group, the level of O-glycosylation was significantly increased and the phosphorylation level was significantly decreased in NAG-Ae treatment. The neurofilament fibers were long filamentous and the proportion of assembly was higher. These results suggest that the changes of O-glycosylation of neurofilament have a certain effect on the assembly of neurofilament. Further in vitro, we only changed the glycosylation of NFs but did not change its phosphorylation level. The results suggest that the effect of deglycosylation alone on assembly is not obvious. Conclusion: the changes of Oglycosylation in vivo affect the phosphorylation of NFs, and the decrease of Oglycosylation may affect the structure and function of NFs by affecting its phosphorylation and neurofilament assembly, resulting in the degeneration of AD nerve cells. The results provide new ideas for the theoretical study of pathogenesis of AD and the development of drugs.
【學(xué)位授予單位】：天津醫(yī)科大學(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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本文編號(hào)：2429147