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应用分子生物学方法确定同期性多灶性肺癌的克隆来源 ,为肺癌的分期提供依据。方法 :对 2例肺部同期多灶性肺癌患者的每个病灶都用石蜡切片做显微解剖及蛋白酶K消化 ,提取基因组DNA ,先后两次PCR扩增K -ras第 1外显子和 p5 3外显子 5～ 9,用微型凝胶做非同位素性SSCP电泳 ,银染色后观察。对有变异电泳图型的样本用DNA序列测定去证实点突变。结果 :例 1的两个肺癌结节 ,仅 1个表现K -ras第 1外显子 12密码子突变 (GGT→TGT) ;例 2的 3个肺癌结节 ,1个表现K -ras第 1外显子 12密码子突变 (GGT→TGT) ,另 1个表现 p5 3第 5外显子 48密码子突变 (GTG→TTG) ,第 3个无突变发现。结论 :此 2例的多灶性肺癌均为不同的克隆来源。因此 ,提出应用非同位素PCR -SSCP分析 ,检测K -ras和p5 3点突变是鉴别多发性肺癌克隆起源的有效方法。
Molecular biology methods to determine the origin of clonal origin of multi-focal lung cancer, provide the basis for the staging of lung cancer. METHODS: Two lesions of lung cancer with multifocal lung cancer were dissected with paraffin sections and digested with proteinase K, and genomic DNA was extracted. The K-ras exon 1 and p5 3 exons 5 to 9, with a micro-gel for non-isotopic SSCP electrophoresis, silver staining observed. Samples of mutated electrophoresis patterns were confirmed by DNA sequencing to confirm point mutations. Results: Only one of the two lung cancer nodules of Example 1 showed a K-ras exon 12 codon mutation (GGT → TGT); three of the lung cancer nodules of Example 2, one of which showed K-ras first Exon 12 codon mutation (GGT → TGT), and the other showed a mutation of codon 5 of exon 5 of p5 3 (GTG → TTG). No mutation was found in the third one. Conclusion: The two cases of multifocal lung cancer are different sources of cloning. Therefore, the application of non-isotope PCR-SSCP analysis to detect K-ras and p5 3 point mutations is an effective method to identify the origin of multiple lung cancer clones.