原发性肝细胞性肝癌中p16基因的失活与P16蛋白表达的研究

作者分别采用多重PCR、SSCP以及以PCR为基础的甲基化分析法，对２５例原发性HCC标本及其相应的非肿瘤肝组织标本中p16基因的缺失、点突变及甲基化情况进行了检测。另用免疫组化SLAB法对３５例（包括前述已作基因检测的２５例）原发性HCC肿瘤标本及其相应的非肿瘤组织标本中P16蛋白的表达情况进行了检测。结果：２５例原发性HCC肿瘤标本中，３例有p16基因缺失，无p16基因缺失的２２例肿瘤标本中均未发现有p16基因点突变存在。肿瘤标本中基因的甲基化发生率为24%（6/25），而全部非肿瘤组织均未出现p16基因甲基化。在３５例HCC肿瘤标本中共发现P16蛋白表达缺失16例(45.7%)，而全部非瘤组织P16蛋白均表达阳性。由此提示，p16基因的失活与P16蛋白表达的丧失与原发性肝细胞性肝癌的发生及发展有关。

A Study On the Inactivation of p16 Genes and the Expressi on of P16 Protein in Primary Hepatocellular Carcinomas

The aim of the present study was to determine wheth er p16 gene is involved in the genesis of primary hepatocellular carcinoma (HCC). Twentyfive primary HCC tumor and corresponding nontumor liver tissue specimens were examined for P16 gene alterations.The identification of deletion of p16 gene exon 1 and exon 2 was performed using comparative multiplex polymerase chain reaction (PCR) analysis. The point mutation of p16 gene exon 2 was investigated by single strand conforma tional polymorphism (SSCP) analysis, and the status of p16 gene methylation was screened using PCRbased methylation analysis. Moreover, 35 parafin specimens of pr imary HCCs with corresponding nontumor liver tissues, including the 25 cases d escribed above for screening of p16 gene alterations, were investigated also for P16 protein expression by using immunohistochemical analysis. The results of comparative multiplex PCR analysis showed that 12% (3/25) p16 gene deletions, including homozygous deletions in 2 tumors and hemizygous deletion in 1 tumor,were found in the primary HCCs samples. No point mutation was identified in the remaining 22 tumor samp les without p16 gene deletions by using SSCP analysis. Hypermethylation was dete cted in 24% (6/25) of tumor samples by PCRbased analysis. However the correspondi ng nontumor liver tissue specimens were always unmethylated at p16 locus. Loss of P16 protein expression, detected by immunohistochemistry, occurred in 16 of 35 (45.7%) tumor samples, whereas all the nontumor liver tissue specimens showed positive p16 staining. These results indicate that inactivation of p16 gene is r elevant to the genesis of HCC.