【作者】 鲁文胜；

【导师】 汪渊；

【作者基本信息】 安徽医科大学 ， 生物化学与分子生物学， 2003， 硕士

【摘要】 目的 对25例膀胱移性细胞癌(bladder transitional cell carcinoma简称膀胱癌bladder cancer)患者的p16基因第一、第二外显子纯合性缺失，第二外显子的点突变，第一外显子区5′CpG岛的甲基化情况及p16蛋白表达进行检测，探讨p16基因在膀胱癌中失活的方式及p16蛋白表达与膀胱癌之间的关系。方法 取患者膀胱癌组织，用酚／氯仿法提取癌组织基因组DNA，设计p16基因第一、二外显子引物，采用聚合酶链反应(polymerase chain reaction，PCR)、PCR-单链构象多态性(PCR-single strand conformational polymorphism，PCR-SSCP)、甲基化敏感限制性内切酶-PCR分析方法，对25例膀胱癌患者的癌组织基因组DNA的p16基因第一、二外显子的纯合性缺失、第二外显子点突变及第一外显子区5′CpG岛的甲基化情况进行分析。应用S-P免疫组织化学方法检测25例膀胱癌和6例正常膀胱粘膜组织中p16基因的表达情况，并分析其意义。结果 在这25例膀胱癌患者中，发现p16基因第二外显子缺失者有4例，第一外显子和第二外显子共同缺失者有1例，第一外显子缺失者1例，p16基因的缺失率为24％(6／25)；未发现p16基因第二外显子的点突变；p16基因第一外显子区5′CpG岛的甲基化率为69.6％(16／23)。p16基因表达在膀胱癌组织中阳性率为52％(13／25)，6例正常膀胱粘膜组织中均阳性，两者比较差异有显著意义(P＜0.01)。随病理分级、临床分期的上升和预后的不良，p16基因的阳性表达率有下降趋势，但差异无统计学意义(P＞0.05)。结论 p16基因的失活方式主要有三种：纯合性缺失、点突变和5′CpG岛的甲基化，但外显子2的纯合性缺失在安徽区域发生的膀胱癌中可能不是一个较频繁发生的“事件”；通过p16基因第一外显子区5′CpG岛异常甲基化失活可能是膀胱癌p16基因失活的主要机制。在这25例膀胱安徽医科大学硕士学位论文鲁文胜·2003年癌患者中，未发现p16基因的点突变，但仍不能排除这种失活机制，我们将通过扩大样本，进一步研究p16基因的点突变情况。p16基因表达产物与膀胱癌的发生发展及预后可能有关系，也有待于扩大例数进一步研究。p16基因表达产物可作为诊断膀胱癌的指标之一。更多还原

【Abstract】 Objective To investigate the inactivation mechanism of p16 gene in bladder cancer and the relationship between p16 protein expression and bladder cancer. The homozygous deletion of p16 gene exon 1 and 2, the point mutation of exon2 and the methylation in exon 1 5’ CpG islands of p16 gene and p16 protein expression in 25 bladder transitional cell carcinoma(bladder cancer) patients were examined. Methods To get tissue samples from 25 bladder cancer patients and extract DNA from tissue samples using phenol/chloroform method, design the primers for detecting the exonl and exon2 of p16 gene. To analyse the deletions of exonl and exon2 and point mutations of exon2 and methylation variations in exon 1 5’ CpG islands of p16 gene by PCR(polymerase chain reaction), PCR-SSCP(PCR-single strand conformational polymorphism) and methylation-sensitive restriction enzyme hydrolysis PCR methods. The expression of p16 gene was determined by S-P immunohistochemical method in 25 specimens of bladder cancer and 6 cases of normal bladder mucosa tissue.Results The p16 gene deletions were found in six samples,four cases had deletion in exon2,one case had deletion in exonl and one in both exonl and exon2 in 25 bladder cancer samples. The homozygous deletion rate of p16 exon2 was 24% (6/25) ; no point mutation of p16 exon2 was found; the methylation rate of the exon 1 5’ CpG islands of p16 gene was 69.6%(16/23). The positive rate of p16 gene expression was 52% (13 of 25) in bladder cancer and 100% (6/6) in all 6 normal bladder mucosae,significantly lower in bladder cancer than in normal tissue (P<0.01). The positive expression rate of p16 gene showed a tendency to descend with the progression of the pathological grade,clinical stage and poorer prognosis,but there was not a statistically significantdifference(P>0.05). Conclusions There are three ways of inactivation mechanism of p16 gene in bladder cancer: homozygous deletion, point mutation and 5’ CpG island methylation. The deletion of p1 6 exon 2 was not a frequent event in Anhui bladder cancer patiens; a statistically significant association was also noticed between p16 gene exon 1 5’ CpG island methylation and the inactivation of p1 6 gene in bladder cancer. Although no point mutation of p16 gene is found in these 25 bladder cancer patients, we can’t conclude that it is not one of the inactivation mechanism and will make further study of the point mutation of p16 by enlarging the samples. There may be a correlation between p16 protein expression and the occurrence and development and prognosis of bladder cancer, p1 6 protein expression will be made further study by enlarging the samples,too.p16 expression protein may be one of the indicators of diagnosis of bladder cancer.更多还原