マイクロRNAの解析と発現ベクターの開発

MicroRNA分析和表达载体开发

• 批准号: 04F04866
• 负责人: 藁科 知子 (桑原 知子) (2006)
• 金额: $ 1.34万
• 依托单位: National Institute of Advanced Industrial Science and Technology
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for JSPS Fellows
• 财政年份: 2004
• 资助国家: 日本
• 起止时间: 2004 至 2006
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-04F04866/
• 关键词: siRNA; レンチウイルス; マイクロインジェクション; 神経; 海馬; 脳; GFP; 脳関門; RNAi; miRNA

siRNAやmiRNAなど、小さな機能性RNAが通常の神経新生や神経幹細胞の細胞増殖、成熟ニューロンへの分化誘導過程などとどのように関係しているのか、いくつかの報告が出ているが、その詳細な分子機構はまだ明らかになっていない。本研究では、脳神経領域で効率よく安定した機能性RNAの研究を行うために、機能性RNAのデリバリー系の開発と、その検討を行った。動物にsiRNAを導入するためにレンチウイルスコンストラクトを作成し、機能性RNA発現カセットが導入された感染細胞を識別するために、マーカーとしてGFPをともに発現する様に設計した。分化に影響を与えない緩衝液で懸濁され濃縮したレンチウイルス液をコントロールとし、siRNAを発現するレンチウイルスコンストラクトを、直接マイクロインジェクションによって神経新生が頻繁に起きる脳海馬領域に導入した。インジェクション後2ヶ月経過後、脳を抽出し海馬領域の切片を作成し、siRNAが与える影響を各分化経路のマーカーとなる神経遺伝子やグリア系遺伝子の多重染色を行い、評価した。この方法での導入デリバリー法とは別に、blood brain barrierを通過できるような薬剤(1.6Mアラビノース)の動物への導入法も検討した。頸動脈を取り出し、カテーテルで固定後アラビノースを注射、一定時間経過後レンチウイルス液を固定カテーテルから注入する。3週間経過後、脳を抽出し海馬領域の切片を作成し、siRNAが与える影響を各分化経路のマーカーとなる神経遺伝子やグリア系遺伝子の多重染色を行い、評価した。これらの結果から、脳に直接マイクロインジェクションを行った方法では、非常に効果的なGFPマーカーの発現と、表的遺伝子のノックダウンによる神経新生阻害現象が、インジェクション箇所である海馬Dantate Gyrus領域に確認できた。本研究成果をもとに、論文投稿の準備を現在進めている。

SiRNA miRNA, small-scale functional RNA are usually used to induce cell colonization and differentiation of mature cells. The process of differentiation is to determine whether or not they are required to report, and that the molecular machinery is responsible for the diagnosis. In this study, in the field of research, the rate of stability and stability in the field of research, the study of functional RNA, the system of functional RNA, the rate of operation, the rate of stability, the degree of stability, the rate of stability, the level of stability, the performance of functional RNA. The siRNA is imported into the system, and the functional RNA is used to detect the infection in the infected cells. In addition, the functional RNA is responsible for the detection of the infection status, the detection of the infection and the monitoring of the GFP. The difference between the two is different from each other. The difference between the two groups is that there is no difference between the two groups. The difference between the two groups is that there is a difference between the two groups. The difference between the two groups is that there is a difference between the two groups. The difference is that there is a difference between the two groups. The difference between the two groups is that there is a difference between the two groups. The difference between the two groups is that there is a difference between the two groups. The difference between the two groups is due to the fact that there is a difference between the two groups. The difference between the two groups is due to the fact that the sea field is not available. At the end of the second month, the sea field sections were taken out of the sea to make them, siRNA and so on. All the differentiation pathways were divided into two different pathways. The method is used to determine the difference between the two methods, and the blood brain barrier method is used to introduce the animals into the law through the monitoring system (1.6 MB). After the device is fixed, it should be injected immediately, and after a certain period of time, the liquid should be fixed and injected into the machine. 3. After the treatment, the sea area was sliced and made, siRNA and DNA were divided into different pathways. The results showed that there was no significant difference between the two groups. The results are as follows: the results, the results The results of this study are ready to be submitted and are now being reviewed.