可分泌Aβ阻断肽的重组腺相关病毒的神经保护作用研究
发布时间：2018-08-29 19:42
【摘要】：阿尔茨海默病（Alzheimer's disease,AD）是常见的神经系统变性疾病，其病理特征为老年炎性斑（senile plaques, SP）、神经原纤维缠结（neurofibrillary tangles,NFTs）、海马锥体细胞颗粒空泡变性(granulovacuolar degeneration)及神经元缺失。临床特征为隐袭起病，缓慢进展，逐渐加重的智能衰退，多伴有人格改变。流行病学研究证实，痴呆业已成为发达国家仅次于心脏病、肿瘤、中风的第4位死因，而AD约占痴呆总数的60%以上。目前全世界约有3560万人患有AD或其他形式的痴呆。在2010年，全球用于治疗痴呆的总花费约为6040亿美元，约相当于全世界国民生产总值的1%。由此可见，阿尔茨海默病不仅给患者本人及其家庭造成巨大的痛苦和经济负担，也已成为一个全球性的社会问题。因此，探寻有效的AD治疗方法，具有非常重要的医学意义和社会学意义，是神经病学研究亟待解决的关键问题，也是每一名神经科学工作者面临的巨大挑战。 近年来，线粒体功能障碍(Mitochondrial dysfunction)对阿尔茨海默病等中枢神经系统退行性疾病的影响日益引起科研人员的关注。Aβ结合乙醇脱氢酶（Aβbinding alcohol dehydrogenase, ABAD）是一种位于线粒体基质内的乙醇脱氢酶，研究发现，当ABAD与Aβ结合时，能够干扰细胞内正常的氧化还原过程，导致线粒体肿胀，神经元死亡。而其阻断肽ABAD-DP能竞争性抑制Aβ与ABAD结合，保护线粒体膜的氧化还原机能，进而拮抗Aβ对神经细胞的毒性作用，因此在AD的治疗中具有巨大的开发潜力。但由于其在体内快速降解并且不能通过血脑屏障和细胞膜，临床难以应用。基于以上原因，本研究首先应用基因工程技术，将ABAD-DP亚克隆入含有信号肽和蛋白质转导域的载体质粒中，构建可携带融合基因的重组腺相关病毒rAAV/NT4-TAT-6His-ABAD-DP（rAAV/NTA）；进一步，通过对体外β-淀粉样肽和过氧化氢的细胞学研究证实可分泌表达ABAD-DP的重组腺相关病毒具有与Aβ42相结合的能力和较强的神经保护作用；最后，利用滴鼻给药的方法验证融合ABAD-DP的重组腺相关病毒在动物水平的神经保护作用和“鼻-脑通路”的存在，为阿尔茨海默病的治疗提供新的思路。 主要研究结果：（1）琼脂糖凝胶电泳和基因测序结果证实融合基因成功插入载体质粒，成功构建了重组腺相关病毒载体pSSGH/NT4-TAT-6His-ABAD-DP，并成功包装出浓度较高的重组腺相关病毒rAAV/NTA；（2）重组腺相关病毒rAAV/NTA能够高效转染Hela细胞，实现融合基因ABAD-DP在细胞内的表达，并成功证实了其与细胞内Aβ42结合的能力；（3）MTT和流式细胞检测结果表明在H2O2的氧化损伤细胞模型中，表达ABAD-DP的重组腺相关病毒能够明显增加细胞数量，改善其活力，减少细胞的凋亡和坏死，具有非特异性的抗氧化应激作用和细胞保护作用；（4）Morris水迷宫实验检测结果表明，经鼻给予可分泌表达ABAD-DP的重组腺相关病毒能够显著改善AD转基因小鼠的记忆能力。 结论与创新：（1）首次成功构建了具有信号肽、蛋白质转导域和ABAD-DP融合基因(NTA)的重组腺相关病毒载体，并成功包装出具有较高滴度的重组病毒rAAV；（2）证实了该重组病毒能够在体外实现表达，并且具有同细胞内Aβ42相结合的能力，间接证实了细胞内Aβ中毒学说的合理性；（3）证实了该重组病毒对过氧化氢诱导的SH-SY5Y细胞具有保护作用。提出可分泌表达ABAD-DP的重组腺相关病毒系统可能不仅仅具有通过拮抗Aβ发挥线粒体保护作用和神经保护作用，还具有非特异性的抗氧化应激作用；（4）通过动物行为学实验证实了该重组病毒可提高阿尔茨海默病转基因动物模型APP695小鼠的记忆能力，发挥神经保护作用。研究结果提示通过经鼻给予腺相关病毒介导的基因药物，是中枢神经系统变性疾病有希望的治疗策略。 总之，本研究通过对线粒体酶的基因改造，提出了一种基因治疗中枢神经系统病的新方法，，这将给具有治疗意义的肽类物质成功到达脑组织靶点发挥生物学作用提供新的思路，为中枢神经系统疾病（如阿尔茨海默病）的治疗带来新的希望。
[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.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R749.16
本文编号：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.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R749.16
【引证文献】
相关博士学位论文 前1条
1 王旭;Aβ相关乙醇脱氢酶阻断肽适配子对Aβ细胞内毒性拮抗作用的研究[D];吉林大学;2013年
本文编号：2212220
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