【摘要】：阿爾茨海默�。ˋlzheimer's disease,AD）是常見的神經系統變性疾病，其病理特征為老年炎性斑（senile plaques, SP）、神經原纖維纏結（neurofibrillary tangles,NFTs）、海馬錐體細胞顆�？张葑冃�(granulovacuolar degeneration)及神經元缺失。臨床特征為隱襲起病，緩慢進展，逐漸加重的智能衰退，多伴有人格改變。流行病學研究證實，癡呆業(yè)已成為發(fā)達國家僅次于心臟病、腫瘤、中風的第4位死因，而AD約占癡呆總數的60%以上。目前全世界約有3560萬人患有AD或其他形式的癡呆。在2010年，全球用于治療癡呆的總花費約為6040億美元，約相當于全世界國民生產總值的1%。由此可見，阿爾茨海默病不僅給患者本人及其家庭造成巨大的痛苦和經濟負擔，也已成為一個全球性的社會問題。因此，探尋有效的AD治療方法，具有非常重要的醫(yī)學意義和社會學意義，是神經病學研究亟待解決的關鍵問題，也是每一名神經科學工作者面臨的巨大挑戰(zhàn)。 近年來，線粒體功能障礙(Mitochondrial dysfunction)對阿爾茨海默病等中樞神經系統退行性疾病的影響日益引起科研人員的關注。Aβ結合乙醇脫氫酶（Aβbinding alcohol dehydrogenase, ABAD）是一種位于線粒體基質內的乙醇脫氫酶，研究發(fā)現，當ABAD與Aβ結合時，能夠干擾細胞內正常的氧化還原過程，導致線粒體腫脹，神經元死亡。而其阻斷肽ABAD-DP能競爭性抑制Aβ與ABAD結合，保護線粒體膜的氧化還原機能，進而拮抗Aβ對神經細胞的毒性作用，因此在AD的治療中具有巨大的開發(fā)潛力。但由于其在體內快速降解并且不能通過血腦屏障和細胞膜，臨床難以應用�；谝陨显�，本研究首先應用基因工程技術，將ABAD-DP亞克隆入含有信號肽和蛋白質轉導域的載體質粒中，構建可攜帶融合基因的重組腺相關病毒rAAV/NT4-TAT-6His-ABAD-DP（rAAV/NTA）；進一步，通過對體外β-淀粉樣肽和過氧化氫的細胞學研究證實可分泌表達ABAD-DP的重組腺相關病毒具有與Aβ42相結合的能力和較強的神經保護作用；最后，利用滴鼻給藥的方法驗證融合ABAD-DP的重組腺相關病毒在動物水平的神經保護作用和“鼻-腦通路”的存在，為阿爾茨海默病的治療提供新的思路。 主要研究結果：（1）瓊脂糖凝膠電泳和基因測序結果證實融合基因成功插入載體質粒，成功構建了重組腺相關病毒載體pSSGH/NT4-TAT-6His-ABAD-DP，并成功包裝出濃度較高的重組腺相關病毒rAAV/NTA；（2）重組腺相關病毒rAAV/NTA能夠高效轉染Hela細胞，實現融合基因ABAD-DP在細胞內的表達，并成功證實了其與細胞內Aβ42結合的能力；（3）MTT和流式細胞檢測結果表明在H2O2的氧化損傷細胞模型中，表達ABAD-DP的重組腺相關病毒能夠明顯增加細胞數量，改善其活力，減少細胞的凋亡和壞死，具有非特異性的抗氧化應激作用和細胞保護作用；（4）Morris水迷宮實驗檢測結果表明，經鼻給予可分泌表達ABAD-DP的重組腺相關病毒能夠顯著改善AD轉基因小鼠的記憶能力。 結論與創(chuàng)新：（1）首次成功構建了具有信號肽、蛋白質轉導域和ABAD-DP融合基因(NTA)的重組腺相關病毒載體，并成功包裝出具有較高滴度的重組病毒rAAV；（2）證實了該重組病毒能夠在體外實現表達，并且具有同細胞內Aβ42相結合的能力，間接證實了細胞內Aβ中毒學說的合理性；（3）證實了該重組病毒對過氧化氫誘導的SH-SY5Y細胞具有保護作用。提出可分泌表達ABAD-DP的重組腺相關病毒系統可能不僅僅具有通過拮抗Aβ發(fā)揮線粒體保護作用和神經保護作用，還具有非特異性的抗氧化應激作用；（4）通過動物行為學實驗證實了該重組病毒可提高阿爾茨海默病轉基因動物模型APP695小鼠的記憶能力，發(fā)揮神經保護作用。研究結果提示通過經鼻給予腺相關病毒介導的基因藥物，是中樞神經系統變性疾病有希望的治療策略。 總之，本研究通過對線粒體酶的基因改造，提出了一種基因治療中樞神經系統病的新方法，，這將給具有治療意義的肽類物質成功到達腦組織靶點發(fā)揮生物學作用提供新的思路，為中樞神經系統疾�。ㄈ绨柎暮Ｄ。┑闹委煄硇碌南Ｍ�。
[Abstract]:Alzheimer's disease (AD) is a common neurodegenerative disease characterized by senile plaques (SP), neurofibrillary tangles (NFTs), granulo-vacuolar degeneration of hippocampal pyramidal cells and neuronal deficits. Epidemiological studies have confirmed that dementia has become the fourth leading cause of death in developed countries after heart disease, cancer and stroke, while AD accounts for more than 60% of the total number of dementia. There are about 35.6 million people worldwide suffering from AD or other forms of dementia. In 2010, dementia was treated globally. Alzheimer's disease has not only caused tremendous pain and economic burden to the patients themselves and their families, but also become a global social problem. Therefore, it is of great medical significance to explore effective treatment for AD. Sociological significance is the key problem to be solved urgently in neurological research, and also a great challenge to every neuroscientist.
In recent years, the effects of mitochondrial dysfunction on degenerative diseases of the central nervous system, such as Alzheimer's disease, have attracted increasing attention of researchers. Beta binding can interfere with the normal redox process in cells, resulting in mitochondrial swelling and neuronal death. Its blocking peptide ABAD-DP can competently inhibit the binding of A beta to ABAD, protect the redox function of mitochondrial membrane, and then antagonize the toxicity of A beta to nerve cells. Therefore, it has great potential in the treatment of AD. For these reasons, ABAD-DP was subcloned into vector plasmid containing signal peptide and protein transduction domain to construct recombinant adeno-associated virus rAAV/NT4-TAT-6His-ABAD-DP with fusion gene. DP (rAAV/NTA); furthermore, cytological studies of beta-amyloid peptide and hydrogen peroxide in vitro confirmed that the recombinant adeno-associated virus secreting and expressing ABAD-DP had the ability to bind to Abet42 and had strong neuroprotective effect; finally, the recombinant adeno-associated virus fused with ABAD-DP was verified at animal level by intranasal administration. Neuroprotective effects and the existence of "nose-brain pathway" provide new ideas for the treatment of Alzheimer's disease.
The main results were as follows: (1) Agarose gel electrophoresis and gene sequencing confirmed that the fusion gene was successfully inserted into the vector plasmid, and the recombinant adeno-associated virus vector pSSGH/NT4-TAT-6His-ABAD-DP was successfully constructed, and the recombinant adeno-associated virus rAAV/NTA with high concentration was successfully packaged; (2) the recombinant adeno-associated virus rAAV/NTA could efficiently transfect Hela. (3) MTT and flow cytometry showed that the recombinant adeno-associated virus expressing ABAD-DP could significantly increase the number of cells, improve their viability, reduce cell apoptosis and deterioration in the oxidative damage cell model induced by H2O2. Morris water maze test showed that recombinant adeno-associated virus secreting ABAD-DP could significantly improve the memory of AD transgenic mice.
Conclusion and Innovation: (1) The recombinant adeno-associated virus vector with signal peptide, protein transduction domain and A BAD-DP fusion gene (NTA) was successfully constructed for the first time, and the recombinant virus rAAV with high titer was successfully packaged; (2) The recombinant virus was confirmed to be able to express in vitro and combine with intracellular Abeta 42. It was suggested that the recombinant adeno-associated virus system secreting and expressing ABAD-DP might not only exert mitochondrial and neuroprotective effects by antagonizing A-beta, but also have nonspecific effects. (4) The recombinant virus can improve the memory ability of transgenic mice with Alzheimer's disease and play a neuroprotective role. The results suggest that adeno-associated virus-mediated gene therapy through nasal administration may be a promising treatment for degenerative diseases of the central nervous system. Therapeutic strategies.
In summary, this study proposes a new method of gene therapy for central nervous system diseases by gene modification of mitochondrial enzymes, which will provide new ideas for the successful arrival of therapeutic peptides at brain tissue targets and play a biological role, and bring new hope for the treatment of central nervous system diseases such as Alzheimer's disease. Look.
【學位授予單位】：吉林大學
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R749.16

【引證文獻】

相關博士學位論文 前1條

1 王旭;Aβ相關乙醇脫氫酶阻斷肽適配子對Aβ細胞內毒性拮抗作用的研究[D];吉林大學;2013年

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