【作者】 雷冬梅；

【导师】 李晓文；

【作者基本信息】 郑州大学 ， 人体解剖与组织胚胎学， 2008， 硕士

【摘要】 目的:家族性腺瘤性息肉病（familial adenomatous polyposis,FAP）是一种常染色体延迟显性遗传病,人群中发病率为1/10 000～1/15 000。未经治疗的患者至40岁左右时,癌变的几率可达100%。近年来,由于筛查制度和预防性结肠切除术的开展,FAP死于大肠癌的比例在下降,但死于肠外肿瘤的比例却呈上升趋逝。通过基因诊断降低本病的发病率、死亡率具有重要意义。在APC基因突变阳性的家族中,患者的子代有50%的发病风险,有不少报道认为,APC基因突变位点与FAP的临床表现相关。本文搜集了两个FAP家系,家系中患者除结直肠肿瘤外,可伴发子宫内膜癌、卵巢癌等肠外恶性肿瘤。通过两个家系APC基因E₁～E₁₄突变的检测分析,为FAP的分子遗传学机制和建立监控系统提供理论依据。肿瘤的发生是多因素、多步骤的复杂过程,是癌基因的激活与多个抑癌基因失活共同作用的结果。与APC同样做为抑癌基因的p53基因在结直肠癌中表达的研究,也越来越受到重视,除了对肿瘤的预后提供指导,也可与FAP基因联合做为筛查的目标之一。本文同时采用SP免疫组化方法对两个FAP家系中结直肠癌患者p53基因的表达进行观察分析,探讨其在结直肠癌生长、浸润、转移中的作用,为临床预测肿瘤的转移潜能和预后提供依据。材料及方法1.2006年至2007年2月,我们共发现2个经临床确诊的FAP家系。2个家系共有成员44人,FAP患者14人。获知情同意后,我们抽取9例患者和24例表型正常的血缘亲属为实验组,1例散发FAP患者和2例随机健康人作为对照组,两组均为汉族,性别、年龄相匹配。提取受试者基因组DNA,PCR-SSCP法检测APC基因第1～14外显子突变,对可疑异常条带进行DNA测序分析。2.收集两个家系FAP患者手术切除结、直肠癌标本20例,绒毛状腺瘤组织5例,绒毛状腺瘤伴不典型增生15例,正常对照15例,采用免疫组织化学S-P法,鼠抗人单克隆抗体p53（MAB-0226）与SP试剂盒均为美国Maxin公司产品（购自福州迈新生物技术公司）,染色设对照组,以PBS代替一抗作阴性对照,用已知阳性切片做阳性对照。观察p53的表达水平。结果:1.经系谱分析,2个家系中FAP呈常染色体显性遗传,并有遗传早现。2.PCR-SSCP电泳显示,DNA条带未发现明显异常。对扩增产物随机抽样进行测序分析,并与APC基因标准cDNA序列（GenBank #NM-000038）进行比对,证实无突变。3.p53基因在FAP患者结、直肠癌中表达强阳性15例;在绒毛状腺瘤伴不典型增生中表达阳性3例;在绒毛状腺瘤中表达阳性6例;在正常结、直肠黏膜中均未见表达。经统计学分析,p53在FAP患者肿瘤组织中的表达与正常结直肠黏膜中的表达有显著性差异。结论:1.本文两个家系未发现APC基因E₁～E₁₄突变。2.FAP的发生有更复杂的分子机理,因此,APC基因可能不应该做为FAP的唯一诊断基因。3.FAP患者结直肠肿瘤组织与正常组织p53的表达有显著性差异,并随肿瘤的演变过程逐渐增高。该基因对FAP的发展、演变起促进作用,但可能不是导致肿瘤发生的早期事件。更多还原

【Abstract】 Objective:Familial adenomatous polyposis （FAP） is an autonomic delayed dominant syndrome affecting 1:10,000 people. It is due to a mutation of the APC gene on the chromosome 5q. If left untreated, carriers have an almost 100% chance of developing colorectal cancer by age 40 after having multiple （typically 100s to 1000s） of adenomatous polyps. Since the introduction of screening and prophylactic colostomy, colorectal cancer related death has decreased, while non- colorectal cancer related death has increased.In the families of APC gene mutation, the children of patients have 50% suffering rate. For the present study, the gene mutation sites were related with manifestations of AFP. we identified two FAP pedigrees in our patients with uterine and ovarian manifestations and subsequently performed APC mutation assay for E₁～E₁₄,with a view to obtaining a better insight into its genetic mechanism, thus providing a theoretical basis for establishing a surveillance system.Tumorigenesis is a multifactor and multistage process, which involves the activation of ontogenesis and inactivation of tumor suppressor genes. As a tumor suppressor gene, p53 plays an important role in such a process. There is evidence that p53 profiling has a predictive value in prognosis. In order to better understand its role in proliferation, invasion and metastasis in those patients, p53 expression was also analyzed, using SP immunohistochemical technology. However, whether it can be used as a predictor in FAP or not is still to be determined by further research.Materials and methods1. From 2006 to February in 2007, we have discovered 2 FAP pedigrees through clinical definite, there were 44 person and 14 FAP sufferers in these 2 pedigrees. The experimental group included 9 patients and 24 normal phenotype relatives, and control group included 1 diverging FAP patients. Two groups are both Han people and sex, age are matching. Extracting DNA of subjects and detecting the 1～14 extron mutation through PCR-SSCP and then making sequencing analysis. 2. Collecting the paraffin block of 2 FAP pedigrees patients from 2006 to 2007. There were 20 rectal cancer, 10villous adenomas and 5villous adenomas accompanied atypical hyperplasia and 15 normal contrast. The specimens of every group were stained according to the description of SP kit. P53 protein was detected using an immunohistochemical kit according to the convention SP method.Results1. FAP was autonomic dominant inheritance in 2 pedigrees and there were inheritance anticipation.2. PCR-SSCP showing: DNA immigration strap has not discovered obviously abnormality. Random selecting the different sites amplification products of 1～14 extron mutation in patients and control groups, there was no mutation through verifications.3. The expression of p53 in colorectal cancer tissues of FAP patients showed increasing tendency and increased gradually following the development of tumor.Conclusions:1. This text has not discovered the E₁～E₁₄ mutation of APC gene in 2 pedigrees.2. The happening of FAP has more complicated molecule mechanism. So the APC gene should not as the only one gene to diagnose perhaps.3. P53 gene promotes the development of FAP, and can be used diagnosis of FAP through assisting APC gene.更多还原