Assessment of genetic changes in the carcinogenesis of squamous cell lung cancer using in situ PCR.

使用原位 PCR 评估鳞状细胞肺癌致癌过程中的遗传变化。

• 批准号: 07457286
• 负责人: SATO Masami
• 金额: $ 1.66万
• 依托单位: Tohoku University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07457286/
• 关键词: in situ PCR; K-ras; point mutation; PCR; in situ hybridization; direct in situ PCR; indirect in situ PCR; K-RAS; in situ hybridization; 早期肺癌; 発癌; 遺伝子異常; 肺癌; 癌関連遺伝子

In situ PCR is a new technique for the localization of low copy number sequences. We investigated to establish a method for the in situ visualization of a point mutation in K-ras codon 12 by indirect in situ PCR.Twenty-five primers were examined to select mutant-specific primers. Harvested cell lines were fixed and suspended in PCR mixture. Forty cycles of PCR in cell suspension was performed in a thermal cycler using a hot start method. Cells were cytocentrifuged onto slides, and postfixation was performed. The specimens on the slides were then hybridized with a digoxigenin-labeled probe, followed by color reaction. Both Calu-1 (mutated : TGT) and NCI-H460 (wild type : GGT) cells had strong hybridization signals in the nuclei with general primers. But with mutant-specific primers, only Calu-1 cells had hybridization signals. No signal was observed without primers or Taq DNA polymerase. Southern blotting of the seme preparation confirmed desired amplification.We conclude that our indirect in situ PCR method shows the feasibility of in situ identification of single cells carrying point mutations. We also applied direct in situ PCR,but this method failed to detect the point mutation. Huge non-specific incorporation of digoxigenin to several length of DNA fragments caused low specificity of this technique.

原位聚合酶链式反应是一种定位低拷贝数序列的新技术。为建立K-ras基因第12密码子点突变的间接原位聚合酶链式反应检测方法，对25条突变特异性引物进行了筛选。将收获的细胞系固定并悬浮在聚合酶链式反应混合物中。在热循环仪中采用热启动方法进行了40个周期的细胞悬液中的PCR。细胞在玻片上进行细胞离心法，并进行后置固定。玻片上的标本与地高辛标记的探针杂交，然后进行显色反应。CALU-1(突变型：TGT)和NCI-H460(野生型：GGT)细胞均有较强的杂交信号。但用突变型特异的引物，只有CALU-1细胞有杂交信号。未加引物或Taq DNA聚合酶时未观察到信号。SIME制备的Southern blotting证实了预期的扩增。我们得出结论，我们的间接原位PCR方法显示了原位鉴定携带点突变的单细胞的可行性。我们还应用了直接原位聚合酶链式反应，但该方法未能检测到点突变。大量地高辛与几个DNA片段的非特异性掺入导致了该技术的低特异性。

项目成果

Sato, et al.: "Sequential loss of heterozygosity in the progression of squamous cell carcinoma of the lung" British Journal of Cancer. (in press). (1998)

Sato 等人：“肺鳞状细胞癌进展中杂合性的连续丧失”英国癌症杂志。

Sagawa, et al.: "K-ras point mutation occurs in the early 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。”

Sato, et al.: "Sequential loss of heterozygosity in the progression of squamous cell carcinoma of the lung." British Journal of Cancer. (in press). (1998)

Sato 等人：“肺鳞状细胞癌进展过程中杂合性的连续丧失。”

佐藤雅美: "非小細胞肺癌におけるBcl-2蛋白,アポトーシスおよびp53蛋白の免疫組織学的検討" 肺癌. 36・6. 785-790 (1996)

佐藤正美：“非小细胞肺癌中 Bcl-2 蛋白、细胞凋亡和 p53 蛋白的免疫组织学研究”《肺癌》36・6（1996）。

遠藤千顕: "PCR-SSCP法による肺扁平上皮癌組織におけるp53遺伝子異常の検出" 日本臨床細胞学会雑誌. 34. 366 (1995)

Chiaki Endo：“通过PCR-SSCP法检测肺鳞状细胞癌组织中的p53基因异常”日本临床细胞学会杂志34. 366(1995)。