【作者】 李春阳；

【导师】 吴逸明；

【作者基本信息】 郑州大学 ， 劳动卫生与环境卫生学， 2002， 硕士

【摘要】 由于环境的恶化，人们生活方式的改变，恶性肿瘤的发病率有所升高，发病年龄趋于下降。肺癌是人类最常见的恶性肿瘤之一，近年来在我国发病率、死亡率均呈上升趋势，上升幅度居于首位。肺癌发病涉及到一系列基因，如K-ras等癌基因和／或p53、p16、Rb等抑癌基因异常改变的复杂过程。 1996年Ohta等用外显子捕获法首次克隆到一个新的抑癌基因—FHIT(fragile histidine triad FHIT)基因，FHIT基因表达异常多出现在与环境接触器官肿瘤中，被认为与环境致癌因素有关，是环境致癌物作用的靶基因。并含有编码组氨酸三联体的功能区，是将染色体位点不稳定性与肿瘤联系起来的一个分子事件，在许多人类肿瘤组织或细胞系，尤其是与环境接触器官的上皮肿瘤中该基因呈现高郑州大学2002年硕士论文 人肺癌脆性组氨酸三联体基因突变的研究频率的纯合性缺失或异常转录，呈现肿瘤特异性。 人类FHIT基因全长约SMb，属于HIT基因家族，共有”10个外显子，编码一个约 1刁 Kb W，外显子 5～9为编码区，外显子5含有开放性阅读框的蛋氨酸起始密码，外显子8包含编码HIT的结构区。FHIT基因与人类最常见的染色体脆性部位FRA3B重合＊hit蛋白是147个氨基酸组成的分子量为 16．SKDa的多肽，核心部分与酵母 S，pombe（Schlzosaecharo啊cespombe）AP4A（dladenos。nes’，5卜’－pl，p4－tetr－aphosphate）水解酶相似。人类Fhit蛋白是AP3A水解酶，通过使AP3A与AP4A浓度保持在一定比例从而对细胞分化产生影响，与提高机体对致癌因素的耐受性有关。 与经典的抑癌基因不同，FHIT基因在转录水平只出现一个或几个外显子的缺失，有时可见小插入，尚未检测到点突变。本实验在本室动物实验基础上，采用PCRSSCP及DNA测序方法，研究肺癌组织FHIT基因DNA水平变异，为进行循环 DNA中 FHIT基因变异研究及 F HIT基因变异对肺癌预警作用可能性研究提供基础资料。方法 收集肺癌患者门0例）、肺良性病变门例）手术标本，离体 20min内迅速放入液氮保存。应用 DNAClub及OMIGA软件设计分析FHIT基因外显子5特异性引物，采 2 郑州大学2002年硕士论文 人肺癌脆性组氨酸三联体基因突变的研究 用PCR扩增目的基因，应用单链构象多态性分析方法及 DNA序列测定技术检测FHIT基因变异。 结果 3 0例肺癌患者中有 7例出现 FHIT基因变异，阳性率“23．3沁其中鳞癌28石％“；’21），腺癌厂伯），而癌旁组织及 肺良性病变组中均无变异，肺癌组、癌旁组及肺良性病变 组比较差异具有统计学意义中叩刀5）。11期肺癌5／l， IH期肺癌2门1，不同病理分期间未见显著差异中＞0刀5） 不同肺癌组织类型中未见显著差异巾叩刀5） DNA序列测定显示，有两个位点的碱基发生了变异： 188位插入一个 G，227位 T7—C。 结论 1．FHIT基因DNA水平异常与人肺癌有密切关系。 2．DNA序列测定显示FH］［T基因内含子存在变异，该变 异可能是引起FHIT基回RNA异常剪接的主要原因之更多还原

【Abstract】 The incidence of malignant tumor is increasing and the onset age is tending to be younger along with the changes of environment and life-tyle of human being. Lung cancer is a common malignant tumor, the increasing of lung cancer is in the first place. Tumorgenesis of lung cancer is related to multiple gene abnormalities, such as p53, ras, c-fos, c-jun, et al. In 1996,Ohta discovered a new gene-FHIT(fragile histidine triad FHIT)by exon- capture technique. Abnormal FHIT has usually been found in various epithelial cancers. It was reported that this genewas closely associated with the environmental carcinogen. FHIT gene is a focus for the study of environmental carcinogen acted upon.Human FHIT gene, a member of the HIT gene family, spans about 500Kb. It encodes 1.1Kb mRNA, including 10 exons, and exon 5 to 9 is encoding domain of HIT.FHIT gene is located at 3p142, it overlaps with FRA3B,which is the most common fragile site in human chromosome. Fhit protein is consisted of 147 amino acids weighted 16.8Kb. The core of Fhit shows similarity to diadenosine 5’,5 -p1,-p4-tetraphosphate(AP4A)hydrolase from the fission yeast Schizosaecharomycespombe. The Fhit from humans is classified as an dinucleoside 5 -5 -p’-p3-triphos-phate(AP3A)hydrolase, it plays a role in cell differentiation and apoptosis by the ratio of AP3A/AP4A. It was reported that Fhit could strengthen cell s endurance to carcinogens:FHIT gene differs from the classical suppressor gene, abnormalities at transcript level were deletion of one or several exohs, sometimes were insertions, but up to now, point mutation has not been found. In this study, DNA fragment of FHIT was analyzed by using PCR-SSCP andDNA sequencing. The purpose of this study is to provide useful information for mutation in plasm DNA and prewarning of lung cancer. MethodsGenomic DNA was isolated from lung cancer tissues, no-tumorous lung tissues, and benign. Specific primers of FHIT gene exon 5 were designed by using DNAClub and OMIGA software. Polymerase Chain Reaction was used to amplify the target gene PCR-SSCP and silver staining were used to find abnormalities. The positive samples were sequenced by dideoxy method with DNAs sequencer. ResultsAmong 6 of 21 lung squamous cell carcinoma, one of 6 lung adenocarcinomas expressed aberrant FHIT gene. The others were normal. There was significant difference between the group of tumors and the group of non-tumor (p<0.05). There were no significant differences among the different histological types.Seqencing analysis of the altered sample showed that two base pairs were detected to be different from the sequence of GeneBank in two samples. (G inserted after position 188,T toC in position 227). Conclusions1. Aberration of FHIT gene in DNA was associated with lung cancer, it may not be associated with the patient’s histological types.2. Mutation in introns of FHIT gene may be one of the reasons altering RNA splicing.更多还原