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α1A-Caチャンネル遺伝子の変異による脳細胞変性の分子病態

α1A-Ca通道基因突变引起脑细胞变性的分子病理学

基本信息

批准号：
14017028
负责人：
水澤英洋
金额：
$ 4.48万
依托单位：
Tokyo Medical and Dental University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research on Priority Areas
财政年份：
2002
资助国家：
日本
起止时间：
2002 至无数据
项目状态：
已结题

项目摘要

目的及び背景:脊髄小脳失調症6型(SCA6)は、多くの神経変性疾患の中でもその原因遺伝子機能が判明している数少ないものの一つであり病態解明に至る可能性が高く、進行性変性症の他発作性症候も呈するなど多彩な神経障害を来たしその機序の解明は科学的インパクトが大きい。またSCA6は本邦に多くニーズも大きい。我々は第19番染色体連鎖小脳失調症を同定、それがSCA6と同一であることやCAGリピート伸長の病的意義を明らかにし、SCA6のプルキンエ細胞質内封入体を発見、Caチャンネル権能の異常を初めて報告するなど一連の研究を続けている。本研究の目的は、培養細胞ならびに動物モデルを作製してα1A-Caチャンネル遺伝子(CACNA1A)の変異による脳細胞変性と機能障害の分子病態を解明することである。方法:まず、これまで用いていたHEK細胞にてSCA6遺伝子を導入し、細胞死を生じる条件を検索し、そのメカニズムを探る。また、変異lox配列をマウスcacnalaエクソン1に導入したES細胞を単離し、変異lox配列、CAGの異常伸長したヒトCACNA1Aの全長cDNA、およびハイグロイマイシン耐性遺伝子を組み込んだノックインベクターとCreリコンビナーゼの発現ベクターとを同時に導入し、マウスcanalaエクソン1にSCA6変異を持つヒトCACNA1AcDNAを挿入した。予想されるパターンでのcDNAの挿入はサザンハイブリダイゼーションとPCR法により確認した。SCA6の変異CACNA1Aを有するES細胞を多数得て胚盤胞へ移植しSCA6のノックインマウスを得る。結果と考察:HEK細胞にてSCA6遺伝子変異を導入して細胞死を生じさせることに成功し、その時に変異蛋白が断片化していることを明らかにした。また、SCA6のノックインマウスを得ることに成功し、現在症候、チャンネル機能障害、神経病理所見などの表現型の解析を行っている。

Objective and background: the etiology of chiropractic microsomia type 6 (SCA6) and multiple neurotic disorders can be used to identify the cause of disease in a small number of cases, from a small number of symptoms to a high likelihood of disease, and to demonstrate that other sexual symptoms of progressive disorders are colorful disorders in order to understand the science of science. There is a lot of money in the country of SCA6. We have confirmed that the 19th chromosome linkage microcytopenia is the same, and that we know the meaning of the disease in the same SCA6. The meaning of the disease is clear. The meaning of the disease is known. The SCA6 has been sealed into the cell. The Ca has been successfully linked to the first report of the study. The purpose of this study is to develop the molecular pathogens that can block the molecular pathogens. The aim of this study is to develop a cell-based animal model that can block the molecular pathogenicity of the molecular pathogenes.At the end of this study, the aim of this study is to develop a cell-based animal model, which can block the molecular pathogenicity of the molecular pathology. in this study, the aim of this study is to develop the molecular pathogenes.At the same time, the molecular pathology can be blocked by the molecular pathogenes.At the same time, the aim of this study is to develop the molecular pathogenicity. The aim of this study is to induce the molecular pathogenicity and to understand the molecular pathogenes.At the same time, the molecular pathogenicity is blocked. Methods: to investigate the conditions and conditions of HEK cell death, cell death, and cell death by using the following methods. The whole length of the ES cell isolation, the normal lox configuration, the full-length cDNA of the CAG constant elongation CACNA1A, the full length of the CAG cell, the total length of the CACNA1A, the total length of the cell, the total length of the "canala", "1" SCA6, "hold"CACNA1AcDNA", "enter". If you want to use the PCR method to confirm your request, you may want to know that the cDNA is correct. Most of the SCA6 embryos CACNA1A have ES cells, and most of them get the embryo cells transplant SCA6 cells. Results the results showed that the HEK SCA6 fragments were successfully transferred into the dead cells, the cell death cycles were successfully detected, and the protein fragments were amplified by RT-PCR. The symptoms, symptoms, signs and signs of the disease, the symptoms, the symptoms