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目的:调查P16基因是否与原发性肝细胞性肝癌(PHCC)的发生有关。方法:采用多重PCR分析法、单链构象多态性(Single strand conform ation polym orphism ,SSCP)分析法及以PCR为基础的甲基化分析法对25例原发性肝细胞性肝癌(PHCC)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检测。用免疫组织化学法对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调查。结果:25 例HCC肿瘤标本中,3 例有P16基因缺失,其中2例为纯合子缺失,1例为半合子缺失。另发现1例P16基因无缺失者有D9s162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。P16 蛋白表达缺失与肿瘤的病理分级有关,分级高者(Ⅲ、Ⅳ级)比分级低者更易发生(P< 0.01)。在25例作了P16基因检?
Objective: To investigate whether the P16 gene is associated with the development of primary hepatocellular carcinoma (PHCC). METHODS: Twenty-five cases of primary hepatocellular carcinoma (PHCC) were analyzed by multiplex PCR, single strand conformation polymorphism (SSCP) analysis, and PCR-based methylation analysis. P16 gene deletions, point mutations, and methylation were detected in specimens and their corresponding non-tumor liver tissue samples. Immunohistochemistry was used to investigate the expression of P16 protein in primary HCC tumor specimens and their corresponding non-tumor tissue specimens in 35 patients (including the previously described 25 genes tested). Results: Of the 25 HCC tumor specimens, 3 had P16 gene deletions, of which 2 were homozygous and 1 was hemizygous. In addition, it was found that 1 case of P16 gene without deletion had D9s162 deletion. No P16 gene point mutation was found in any of the 22 tumor samples without P16 gene deletion. The methylation rate of genes in tumor samples was 24% (6/25), and all non-tumor tissues did not show methylation of P16 gene. In the 35 HCC tumor specimens, 16 cases (45.7%) of P16 protein were deleted, and all non-tumor tissues were positive for P16 protein. Loss of P16 protein expression was associated with the pathological grade of the tumor. Higher grades (III, IV) were more likely to occur than those with low grade (P < 0.01). P16 gene detection was performed in 25 cases.