控制正常细胞对肺癌细胞P~(16)基因缺失的污染的方法的建立及其应用

目的建立一种能有效提高从混有正常细胞的肿瘤组织中检测Ｐ１６基因纯合缺失的灵敏度的方法，以及探讨该基因的状态与肺癌临床病理的关系。方法将野生型ＤＮＡ与Ｐ１６基因纯合缺失的ＤＮＡ按一定比例混合，并将其有限稀释，以不同量的ＤＮＡ为模板，用聚合酶链反应（ＰＣＲ）扩增Ｐ１６基因第二外显子及内对照基因片断，在此基础上，以特定量肺癌ＤＮＡ为模板，用相同的ＰＣＲ条件对上述片断进行扩增，对扩增出的第二外显子行单链构型多态性（ＳＳＣＰ）分析。结果ＤＮＡ模板为５ｎｇ，野生型ＤＮＡ不超过总ＤＮＡ４０％时，不掩盖Ｐ１６基因纯合缺失。小细胞肺癌，癌旁肺无Ｐ１６基因纯合缺失及突变，５２例非小细胞肺癌中有２１例Ｐ１６基因纯合缺失，３例第二外显子ＳＳＣＰ异常，其中伴淋巴结转移的非小细胞肺癌Ｐ１６基因的纯合缺失率（７２％）显著高于无淋巴结转移者（１６．６％）。同时还发现Ｐ１６基因的异常与非小细胞肺癌的临床病理分级及预后明显相关。Ｐ１６基因缺失的患者术后存活时间显著低于无Ｐ１６基因缺失者。结论控制ＰＣＲ中模板的量有助于提高检测Ｐ１６基因缺失的灵敏度。Ｐ１６基因异常可能参与非小细胞肺癌的恶性进展。

Development and application of the method for controlling contamination of tumor samples with normal cells to detect deletion of P 16 gene in prinary lung carcinoma

Objective The method is to be established to effectively promote the sensitivity of the detection of P 16 gene deletion from tumor tissues mixed with normal cells and to determine the relationship between the gene alteration and the lung cancers.Methods The normal DNA and that of MCF 7 cells with P 16 gene deletion was proportionally mixed and the mixture was limitedly diluted.The exon 2 of P 16 gene and internal control gene fragment were amplified with template of various amounts of DNA. And polymerase chain reaction(PCR)technique was used in the amplification of exon2of P 16 gene with certain amount of lung cancer DNA as the templates.The amplified fragment was analyzed with SSCP.Results Normal DNA did not contaminate P 16 gene deletion when DNA template was 5 ng and normal cells less than 40%.No deletion and mutation of P 16 gene was found in the small cell lung cancer and normal cancer surrounding lung tissues,while P 16 gene deletion and mutation were found respectively in 21 and 3 cases of 52 cases of non small cell lung cancer.The rate of P 16 gene deletion (72%) in lung cancer with lymphatic involment was significantly higher than that in lung cancer without the metastasis (16.8%)( P <0.05).It is found that P 16 gened eletion was markedly associated with pathologic manifestation and prognosis of non small cell lung cancer( P < 0.05). Conclusions Controlling of the amount of template DNA in PCR helps to promote the sensitivity of detection of P 16 gene deletion.The abnormality of P 16 gene might relate to the malignant development of non small cell lung cancer.