可分泌Aβ阻斷肽的重組腺相關(guān)病毒的神經(jīng)保護(hù)作用研究
發(fā)布時(shí)間：2018-08-29 19:42
【摘要】：阿爾茨海默�。ˋlzheimer's disease,AD）是常見(jiàn)的神經(jīng)系統(tǒng)變性疾病，其病理特征為老年炎性斑（senile plaques, SP）、神經(jīng)原纖維纏結(jié)（neurofibrillary tangles,NFTs）、海馬錐體細(xì)胞顆�？张葑冃�(granulovacuolar degeneration)及神經(jīng)元缺失。臨床特征為隱襲起病，緩慢進(jìn)展，逐漸加重的智能衰退，多伴有人格改變。流行病學(xué)研究證實(shí)，癡呆業(yè)已成為發(fā)達(dá)國(guó)家僅次于心臟病、腫瘤、中風(fēng)的第4位死因，而AD約占癡呆總數(shù)的60%以上。目前全世界約有3560萬(wàn)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(fèi)約為6040億美元，約相當(dāng)于全世界國(guó)民生產(chǎn)總值的1%。由此可見(jiàn)，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(jīng)濟(jì)負(fù)擔(dān)，也已成為一個(gè)全球性的社會(huì)問(wèn)題。因此，探尋有效的AD治療方法，具有非常重要的醫(yī)學(xué)意義和社會(huì)學(xué)意義，是神經(jīng)病學(xué)研究亟待解決的關(guān)鍵問(wèn)題，也是每一名神經(jīng)科學(xué)工作者面臨的巨大挑戰(zhàn)。 近年來(lái)，線粒體功能障礙(Mitochondrial dysfunction)對(duì)阿爾茨海默病等中樞神經(jīng)系統(tǒng)退行性疾病的影響日益引起科研人員的關(guān)注。Aβ結(jié)合乙醇脫氫酶（Aβbinding alcohol dehydrogenase, ABAD）是一種位于線粒體基質(zhì)內(nèi)的乙醇脫氫酶，研究發(fā)現(xiàn)，當(dāng)ABAD與Aβ結(jié)合時(shí)，能夠干擾細(xì)胞內(nèi)正常的氧化還原過(guò)程，導(dǎo)致線粒體腫脹，神經(jīng)元死亡。而其阻斷肽ABAD-DP能競(jìng)爭(zhēng)性抑制Aβ與ABAD結(jié)合，保護(hù)線粒體膜的氧化還原機(jī)能，進(jìn)而拮抗Aβ對(duì)神經(jīng)細(xì)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(nèi)快速降解并且不能通過(guò)血腦屏障和細(xì)胞膜，臨床難以應(yīng)用。基于以上原因，本研究首先應(yīng)用基因工程技術(shù)，將ABAD-DP亞克隆入含有信號(hào)肽和蛋白質(zhì)轉(zhuǎn)導(dǎo)域的載體質(zhì)粒中，構(gòu)建可攜帶融合基因的重組腺相關(guān)病毒rAAV/NT4-TAT-6His-ABAD-DP（rAAV/NTA）；進(jìn)一步，通過(guò)對(duì)體外β-淀粉樣肽和過(guò)氧化氫的細(xì)胞學(xué)研究證實(shí)可分泌表達(dá)ABAD-DP的重組腺相關(guān)病毒具有與Aβ42相結(jié)合的能力和較強(qiáng)的神經(jīng)保護(hù)作用；最后，利用滴鼻給藥的方法驗(yàn)證融合ABAD-DP的重組腺相關(guān)病毒在動(dòng)物水平的神經(jīng)保護(hù)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(jié)果：（1）瓊脂糖凝膠電泳和基因測(cè)序結(jié)果證實(shí)融合基因成功插入載體質(zhì)粒，成功構(gòu)建了重組腺相關(guān)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(guān)病毒rAAV/NTA；（2）重組腺相關(guān)病毒rAAV/NTA能夠高效轉(zhuǎn)染Hela細(xì)胞，實(shí)現(xiàn)融合基因ABAD-DP在細(xì)胞內(nèi)的表達(dá)，并成功證實(shí)了其與細(xì)胞內(nèi)Aβ42結(jié)合的能力；（3）MTT和流式細(xì)胞檢測(cè)結(jié)果表明在H2O2的氧化損傷細(xì)胞模型中，表達(dá)ABAD-DP的重組腺相關(guān)病毒能夠明顯增加細(xì)胞數(shù)量，改善其活力，減少細(xì)胞的凋亡和壞死，具有非特異性的抗氧化應(yīng)激作用和細(xì)胞保護(hù)作用；（4）Morris水迷宮實(shí)驗(yàn)檢測(cè)結(jié)果表明，經(jīng)鼻給予可分泌表達(dá)ABAD-DP的重組腺相關(guān)病毒能夠顯著改善AD轉(zhuǎn)基因小鼠的記憶能力。 結(jié)論與創(chuàng)新：（1）首次成功構(gòu)建了具有信號(hào)肽、蛋白質(zhì)轉(zhuǎn)導(dǎo)域和ABAD-DP融合基因(NTA)的重組腺相關(guān)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(shí)了該重組病毒能夠在體外實(shí)現(xiàn)表達(dá)，并且具有同細(xì)胞內(nèi)Aβ42相結(jié)合的能力，間接證實(shí)了細(xì)胞內(nèi)Aβ中毒學(xué)說(shuō)的合理性；（3）證實(shí)了該重組病毒對(duì)過(guò)氧化氫誘導(dǎo)的SH-SY5Y細(xì)胞具有保護(hù)作用。提出可分泌表達(dá)ABAD-DP的重組腺相關(guān)病毒系統(tǒng)可能不僅僅具有通過(guò)拮抗Aβ發(fā)揮線粒體保護(hù)作用和神經(jīng)保護(hù)作用，還具有非特異性的抗氧化應(yīng)激作用；（4）通過(guò)動(dòng)物行為學(xué)實(shí)驗(yàn)證實(shí)了該重組病毒可提高阿爾茨海默病轉(zhuǎn)基因動(dòng)物模型APP695小鼠的記憶能力，發(fā)揮神經(jīng)保護(hù)作用。研究結(jié)果提示通過(guò)經(jīng)鼻給予腺相關(guān)病毒介導(dǎo)的基因藥物，是中樞神經(jīng)系統(tǒng)變性疾病有希望的治療策略。 總之，本研究通過(guò)對(duì)線粒體酶的基因改造，提出了一種基因治療中樞神經(jīng)系統(tǒng)病的新方法，，這將給具有治療意義的肽類物質(zhì)成功到達(dá)腦組織靶點(diǎn)發(fā)揮生物學(xué)作用提供新的思路，為中樞神經(jīng)系統(tǒng)疾�。ㄈ绨柎暮Ｄ。┑闹委煄�(lái)新的希望。
[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(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R749.16
本文編號(hào)：2212220
[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(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R749.16
【引證文獻(xiàn)】
相關(guān)博士學(xué)位論文 前1條
1 王旭;Aβ相關(guān)乙醇脫氫酶阻斷肽適配子對(duì)Aβ細(xì)胞內(nèi)毒性拮抗作用的研究[D];吉林大學(xué);2013年
本文編號(hào)：2212220
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