口腔颊癌p16INK4/CDKN2基因的纯合子缺失与点突变

目的 探讨p16基因纯合子缺失和点突变与颊癌发生、发展的关系。方法 采用聚合酶链反应（PCR）、DNA单链构象多态性（SSCP）分析和DNA测序技术研究30例颊癌及10例颊黏膜白斑p16基因纯合子缺失和点突变的情况，并应用免疫组化检测基因改变组织中P16蛋白质的表达。结果 颊白斑和正常颊黏膜无p16基因的纯合子缺失和点突变。在30例颊癌标本中，7例标本p16纯合子缺失，纯合子缺失率是23.3%（7/30）；5例颊癌出现点突变，均发生在第2外显子上，点突变率为16.7%（5/30）。经DNA直接测序，发现5例点突变均为错义突变，其中3例突变位点相同，均在第99密码子上由GAT突变为AAT，由门冬酰胺取代门冬氨酸；对纯合子缺失和点突变的12例颊癌组织进行P16蛋白的检测，发现11例标本P16蛋白表达缺失，1例点突变标本表达正常。结论 p16基因纯合子缺失和点突变是颊癌基因失活的主要形式，与颊癌的发生、发展密切相关。

Study of p16INK4/CDKN2 Gene Homozygous Deletions and Point Mutation in Squamous Cell Carcinoma of Buccal Mucosa

OBJECTIVE: To explore the correlation between homozygous deletions and mutation of p16 gene and the carcinogenesis and progression of squamous cell carcinoma of buccal mucosa. METHODS: Thirty buccal cancers, 10 leukoplakias and 8 buccal mucosas were involved. DNA was extracted from the tissues. PCR was used to analyses homozygous deletion of p16 gene. PCR-SSCP-DNA sequencing was performed to detect the point mutation of p16 gene. Immunohistochemical techniques were used to detect the expression of P16 protein. RESULTS: Gene deletions and point mutations were not found in leukoplakia and normal buccal mucosa. Gene deletions were found in 7 samples out of 30 cases of squamous cell carcinoma of buccal mucosa (23.3%), while point mutations were found in 5 samples out of 30 cases of squamous cell carcinoma of buccal mucosa (16.7%). Sequencing analysis showed that 5 cases point mutations were missense mutations, occurred on exon 2. Three cases occurred in the same point, codon 99 (GAT --> AAT). The result of immunohistochemical stains showed that 11 out of 12 cases gene inactivation did not expressed P16 protein. CONCLUSION: Homozygous deletion and point mutation of p16 were the main pattern of gene inactivation in squamous cell carcinoma of buccal mucosa. There was a closely correlation between p16 gene inactivation and the carcinogenesis of squamous cell carcinoma of buccal mucosa.