in situ PCR法による肺癌発癌過程における遺伝子異常局在の病理学的な解明

原位PCR法病理学阐明肺癌癌变过程中基因定位异常

• 批准号: 09877254
• 负责人: 藤村 重文
• 金额: $ 1.28万
• 依托单位: Tohoku University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Exploratory Research
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 无数据
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09877254/
• 关键词: in situ PCR; K-ras; 点突然変異; PCR; in situ hybridization

in situ PCR法は,細胞内で目的の遺伝子をPCRの技法を用いて増幅させ,その後in situ hybridizationにより検出するもので,従来検出限界以下だった遺伝子のin situでの同定に期待が持たれている.1細胞あたり1コピーの遺伝子の存在をも顕微鏡下に同定できるこの技術はこれまでの病理診断や発癌過程の推論などを根本から変革してしまう可能性を持っている.また,早期診断や前癌病変の診断等の臨床への応用も幅広く考え得る.今回我々は,肺癌における遺伝子変化,特に従来in situ hybrization法で検出し得なかった点突然変異を、in situ PCR法により細胞1個のレベルで同定することを試みた.in situ PCRは,遺伝子やプライマーが異なれば大きく条件が変わると考えられており,また,その条件が非常に多岐に亘る.本年度の研究によって,標本の固定法(緩衝ホルマリン,パラホルムアルデヒド,エタノール,グルタールアルデヒド),前処置法(プロテイナーゼK,ペプシン),プライマーの選定(ミスマッチプライマー,ダブルミスマッチプライマー),PCRの条件(種々の温度およびサイクル数),シグナルの検出(染色)法(RI標識,ジゴキシゲニン)に加えて,in situ hybridizationの条件(ハイブリ溶液の組成,温度,時間)と後固定法の条件(固定液,時間)を確立することができ,K-ras遺伝子の点突然変異を細胞単位で検出することに成功した.

The in situ PCR method was used to detect the intracellular target, and the PCR technique was used to measure the amplitude of the cell, and then the in situ hybridization method was used to detect the size of the cell. It is expected that below the limit, there will be significant differences between the two groups. 1. There is a significant difference between the two groups in terms of the possibility that there is a significant difference in the process of cancer. In the early stage, before the cancer was cut off, the cancer was cut off, and so on. This time, we are sorry that the lung cancer has become obsolete, so we have come to know that the in situ hybrization method has caused a sudden drop in temperature, and that the in situ PCR method has been used to determine the same level of temperature. In situ PCR, the conditions for lung cancer are very different from each other. In this year's study, this year's research has been conducted in terms of the standard method, the standard method, the pre-setting method, and the PCR condition. The RI identification (staining) method adds the temperature, and the in situ hybridization condition (solution composition, temperature, time) of the post-fixation method (stationary liquid, time) makes sure that the temperature is stable, and the cell position of the K-ras is suddenly activated to verify the success of the test.

项目成果

Sagawa, et al.: "K-ras point mutation occurs in the earyly stage of carcinogenesis in lung cancer." British Journal of Cancer. 77(5). 720-723 (1997)

Sakawa等人：“K-ras点突变发生在肺癌的癌变早期。”

Sagawa, et al.: "Detection of K-ras point mutation by in situ PCR in cell suspensions." Lung Cancer. 17. 181-195 (1997)

Sakawa 等人：“通过细胞悬浮液中的原位 PCR 检测 K-ras 点突变。”