【作者】 楼建军；

【导师】 朱小东；

【作者基本信息】 广西医科大学 ， 肿瘤学， 2012， 博士

【摘要】 背景与目的鼻咽癌是我国的高发肿瘤,也是我区高发肿瘤之一,其病因包括EB病毒的感染、化学致癌因素及遗传易感因素等,然而在同一地区同样的暴露条件下的不同人群并不都患肿瘤,其中必然存在个体差异,即遗传易感性。单核苷酸多态性(Single nucleotide Polymorphisms, SNPs)是人类可遗传的变异中最常见的一种,占所有已知多态性的90%以上,是近年来肿瘤研究热点之一。本项目的主要目的是研究相关抑癌基因的单核苷酸多态性与鼻咽癌的发生发展的关系,为鼻咽癌的诊治提供相关理论基础。选择与鼻咽癌相关的多个抑癌基因,包括p53、p16及Rb基因,通过对鼻咽癌患者与正常对照人群相关抑癌基因重要的SNP位点进行对比研究,期望阐明遗传易感性在鼻咽癌发生发展中的作用,也可以应用于鼻咽癌高危人群的筛选,为鼻咽癌的早期干预提供理论基础。本研究计划对鼻咽癌与正常对照人群p53、p16及Rb基因6个重要的SNP位点进行对比研究,期望阐明相关抑癌基因p53、p16及Rb基因在广西鼻咽癌发生发展过程中的作用及其相互关系,为进一步开发基因芯片实现高通量检测打下基础,对鼻咽癌的诊治提供新的思路,进一步降低鼻咽癌发生率和死亡率,以更好地为广大患者服务。材料与方法1.样本与资料的收集应用病例-对照研究的方法在广西南宁人群中,收集了2010年6月到2011年4月临床资料完整的在广西医科大学附属肿瘤医院放疗科住院患者的血液标本200例,男性100例,女性100例；正常对照组200例：男性100例,女性100例。两组人群血液标本都来源于广西南宁,均为南宁本地居民。鼻咽癌组和对照人群组界定标准：鼻咽癌组：经临床和病理学均确诊为癌,每位患者住院之前均未接受过化学治疗、放射治疗或生物治疗等治疗措施的干预。对照人群组：来源于同期身体健康的志愿献血者。2.实验方法用试剂盒法提取得到血液的DNA,经过定量分析后置于-20℃冰箱保存备用。用聚合酶链式反应-限制性片段长度多态性方法(polymerase chain reaction-restriction fragment length polymorphism, PCR-RFLP)及测序方法分析基因多态性及其基因突变分布频率。结果本实验研究主要对常见的抑癌基因的多态性与鼻咽癌的遗传易感性的关系进行的初步研究,所得的结果如下：1.p53、p16及Rb基因多态性与鼻咽癌遗传易感性的关联分析1.1 p53基因1.1.1在鼻咽癌组与对照人群组p53基因rs 117562731位点携带变异等位基因的频率分别为30.0%(60/200)和8.0%(16/200)。两组之间基因型分布比较,差异有统计学意义(χ2=31.449,P=0.000)。1.1.2在鼻咽癌组与对照人群的p53基因rs 114831472位点携带变异等位基因的频率分别为12.5%(25/200)和7.5%(15/200)。两组之间基因分布比较,差异无统计学意义(χ2=2.778,P=0.096)。1.2 p16基因1.2.1在鼻咽癌组与对照人群的p16基因rs 80235406位点携带变异等位基因的频率分别为26.5%(53/200)和9.0%(18/200)。两组之间基因分布比较,差异有统计学意义(χ2=20.977,P=0.000)。1.2.2在鼻咽癌组与对照人群的p16基因rs 121434309位点携带变异等位基因的频率分别为22.5%(45/200)和8.5%(17/200)。两组之间基因分布比较,差异有统计学意义(χ2=14.965,P=0.000)。1.3 Rb基因1.3.1在鼻咽癌组与对照人群的Rb基因rs 117538467位点携带变异等位基因的频率分别为34.5%(69/200)和11.5%(23/200)。两组之间基因分布比较,差异有统计学意义(χ2=29.870,P=0.000)。1.3.2在鼻咽癌组与对照人群的Rb基因rs 117538467位点携带变异等位基因的频率分别为28.0%(56/200)和12.5%(25/200)。两组之间基因分布比较,差异有统计学意义(χ2=14.877,P=0.000)。2.p53、p16及Rb基因多态性与鼻咽癌临床表型的关联研究2.1与AA基因型比较,p53基因rs114831472位点的GG基因型的频率在T1组中高于T2-4组(7.9% VS 1.2%),差异有显著性,提示GG基因型与局部侵袭(T分期)有显著性关联(p=0.017)。p53基因rs117562731 SNP位点CT基因型的频率与CC基因型频率比较,在M1组中高于Mo组(44.4% VS 15.7%),差异有显著性,提示CT基因型与远处转移(M分期)有显著性关联(p=0.030)。2.2 p16基因rs80235406 SNP位点TT基因型的频率与CC基因型频率比较,在No-1组中明显低于N2-N3组(1.6% VS 9.4%),差异有显著性,说明TT基因型与淋巴结转移(N分期)有显著性关联(p=0.011)。p16基因rs80235406 SNP位点TT基因型的频率与CC基因型频率比较,在Mo组中明显低于M1组(3.1% VS 33.3%),p16基因rs121434309 SNP位点GG基因型的频率与AA基因型频率比较,在M1组中高于Mo组(22.2% VS 2.1%),差异有显著性,说明这两个基因型与远处转移(M分期)均有显著性关联(p=0.000和p=0.001)。2.3 Rb基因rs117538467 SNP位点GG基因型的频率与CC基因型频率比较,在M0组中明显低于M1组(4.2% VS 33.3%),Rb基因rs117209587 SNP位点GG基因型的频率与AA基因型频率比较,在M1组中高于Mo组(22.2% VS 3.1%),差异有显著性,说明这两个基因型与远处转移(M分期)均有显著性关联(p=0.023和p=0.002)。结论1.鼻咽癌组与正常对照人群的p53基因rs 114831472位点基因突变与分布无明显差异,说明p53基因rs 114831472位点基因突变与鼻咽癌的发生无显著相关性。2.鼻咽癌组与正常对照人群的p53基因rs 117562731位点基因突变与分布有明显差异,说明p53基因rs 117562731位点基因突变与鼻咽癌的发生有显著相关性。3.鼻咽癌组与正常对照人群的p16基因rs 80235406位点和rs121434309位点基因突变与分布有明显差异,说明p16基因的两个位点基因突变与鼻咽癌的发生有显著相关性。4.鼻咽癌组与对照人群的Rb基因rs 117538467位点和rs 117538467位点基因突变与分布有明显差异,说明Rb基因的两个位点基因突变与鼻咽癌的发生有显著相关性。5.p53、p16及Rb基因多态性与鼻咽癌临床表型及疾病进展有显著相关性。更多还原

【Abstract】 Background and ObjectivesNasopharyngeal carcinoma is the high incidence of cancer in China and is one of the high incidence of tumors in Guangxi. Nasopharyngeal carcinoma were caused by the Epstein-Barr virus infection, chemical carcinogens and genetic susceptibility factors.However, the different groups of people in the same area and the same exposure conditions are not suffered from cancer, which there are differences in individuals in genetic susceptibility.Single nucleotide polymorphism (SNP) is the most common form of human genetic variation, accounting for more than 90% of all known polymorphisms,which is research focus of the tumor in recent years.The main purpose of this project is study the relations between tumor suppressor gene single nucleotide polymorphisms and nasopharyngeal cancer occurrence and developments,to provide relevant theoretical basis for the diagnosis and treatment of nasopharyngeal carcinoma. Some tumor suppressor genes were selected, including p53, p16 and Rb. Related tumor-suppressor genes SNP site were studied in Nasopharyngeal carcinoma patients and Control group.The role of genetic susceptibility were expected to clarify in nasopharyngeal carcinoma occurrence and development,which can also be used for the screening the high risk group and provide a theoretical basis for early intervention of nasopharyngeal carcinoma.In this study,6 SNP sites of p53, p16 and Rb were studied in Nasopharyngeal carcinoma patients and Control group. The role and the mutual relationship were expected between the related tumor suppressor gene p53,p16 and Rb and Nasopharyngeal carcinoma occurrence and development in Guangxi. To lay the foundation for the further development of gene chips for high-throughput detection,to provide new ideas diagnosis and treatment of nasopharyngeal carcinoma,to further reduce the incidence and mortality of nasopharyngeal carcinoma and in order to better for the patients.Materials and Methods1.Sample and data collection A total of 200 NPC blood samples(100 cases of men and 100 cases of women) were selected from June 2010 to April 2011 in Affiliated Tumor Hospital of Guangxi Medical University, Department of Radiation Oncology,and clinical data were collected. A total of 200 Control group blood samples were selected from healthy voluntary blood donors(100 cases of men and 100 cases of women).The two group population’s blood samples are derived from Nanning,Guangxi. The define standard of Nasopharyngeal carcinoma group and control group:Nasopharyngeal carcinoma groups:are diagnosed by the Clinical and Pathological;each of the patients were not received chemotherapy, radiation therapy or biological treatment of intervention treatment measures before hospitalization. Control group:from the healthy volunteer donors.2. MethadsGenomic DNA were extracted from blood leukocytes of all participants,which be placed in a-20℃refrigerator after quantitative analysis. To observe the gene polymorphism and genetic mutation frequency distribution in the two groups, the SNP site were analysed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and sequencing methods.ResultsThe experimental research focus on the relationship between the common tumor-suppressor gene polymorphism and the genetic susceptibility of nasopharyngeal carcinoma.The results as follows:1. p53、p16 and Rb gene polymorphism and the genetic susceptibility of nasopharyngeal carcinoma1.1 p53 gene1.1.1 Among the NPC group and Control group, the genetic mutation rate of SNP locus rs117562731 of p53 were 20.0%(40/200) and 7.0%(14/200) respectively. The allele and genotype frequency had significant difference between the patients with the normal population(x2=2.933, p=0.402).1.1.2 Among the NPC group and Control group, the genetic mutation rate of SNP locus rs114831472 of p53 gene were 12.5%(25/200) and 7.5%(15/200) respectively. The allele and genotype frequency had no significant difference between the patients with the normal population (χ2=2.933,P=0.402).1.2 p16 gene1.2.1 The Nasopharyngeal carcinoma group and Control group, the genetic mutation rate of SNP site rs80235406 of p16 gene were 26.5% (53/200) and 9.0%(18/200) respectively. The allele and genotype frequency had significant difference between the patients with the normal population(χ2=20.977, P=0.000).1.2.2 The Nasopharyngeal carcinoma group and Control group, the genetic mutation rate of SNP site rs 121434309 of p16 gene were 22.5%(45/200) and 8.5%(17/200) respectively. The allele and genotype frequency had significant difference between the patients with the normal population(χ2=14.965, P=0.000).1.3 Rb gene1.3.1 The Nasopharyngeal carcinoma group and Control group, the genetic mutation rate of SNP site rs 117538467 of Rb gene were 34.5%(69/200) and 11.5%(23/200) respectively. The allele and genotype frequency had significant difference between the patients with the normal population(x2=29.870, P=0.000).1.3.2 The Nasopharyngeal carcinoma group and Control group, the genetic mutation rate of SNP site rs 117538467 of Rb gene were 28.0%(56/200) and 12.5%(25/200)respectively. The allele and genotype frequency had significant difference between the patients with the normal population(χ2=14.877, P=0.000).2. p53、p16 and Rb gene and NPC clinical phenotypesWe analyzed the distribution of p53,p16 and Rb gene genotypes among difference clinical characteristics and assessed the relationship between these gene polymorphism and clinical and characteristics in 200 NPC patients.2.1 We found a significant associated between the p53 gene SNP locus rs114831472 GG genotype frequency and primary tumor T stages. (GG versus AA,7.9% VS 1.2%,p=0.017). The significant associated between the p53 gene SNP locus rs117562731 CT genotype frequency and distant metastasis (M stage). (CT versus CC, 44.4% VS 15.7%,p=0.030).2.2 The p16 gene SNP locus rs80235406 TT polymorphism had significant difference with Lymph node metastasis (N stage)(TT versus CC,1.6% VS 9.4%,p=0.011).The p16 gene SNP locus rs80235406 TT and rs121434309 GG polymorphism had significant difference with distant metastasis (M stage). (TT versus CC,3.1% VS 33.3%,p=0.000; GG versus AA,22.2% VS2.1%,p=0.001).2.3 The Rb gene SNP locus rs117538467 GG and rs117209587 GG polymorphism had significant difference with distant metastasis (M stage). (GG versus CC,4.2% VS 33.3%,p=0.023; GG versus AA,22.2% VS3.1%,p=0.002).Conclusions1. Among the NPC group and Control group, the genetic mutation and distribution of SNP locus rs114831472 of p53 gene had no significant difference between the patients with the normal population. The result indicates that the SNP locus rs114831472 of p53 gene polymorphism is not significantly associated with Nasopharyngeal carcinoma. 2. Among the NPC group and Control group, the genetic mutation and distribution of SNP locus rs117562731 of p53 gene had significant difference between the patients with the normal population. The result indicates that the SNP locus rs117562731 of p53 gene polymorphism is significantly associated with Nasopharyngeal carcinoma.3. Among the NPC group and Control group, the genetic mutation and distribution of SNP locus rs80235406 and rs121434309 of p16 gene had significant difference between the patients and the normal population. The result indicates that the SNP locus rs80235406 and rs 121434309 of pi6 gene polymorphism is significantly associated with Nasopharyngeal carcinoma.4. Among the NPC group and Control group, the genetic mutation and distribution of SNP locus rs117538467 and rs117538467 of Rb gene had significant difference between the patients and the normal population. The result indicates that the SNP locus rs117538467 and rs117538467 of Rb gene polymorphism is significantly associated with Nasopharyngeal carcinoma.5. The p53, p16 and Rb genetic polymorphism had significant difference with clinical phenotype and progression of nasopharyngeal carcinoma.更多还原