【作者】 杨永波；

【导师】 周清华； 陈军； 刘红雨；

【作者基本信息】 天津医科大学 ， 外科学， 2010， 硕士

【摘要】 目的非小细胞肺癌是我国发病最高的恶性肿瘤,由于缺乏有效的早期发现方法,因而确诊时多属晚期,除了手术之外,尚无其他的有效治疗方法。术后5年生存率只有约15%。同其他肿瘤一样,非小细胞肺癌的发生、发展是一个多阶段,受多基因调控的过程,由多种肿瘤相关基因表达失常或肿瘤抑制基因失活所致。因此要了解癌变过程中的基因变化,需要研究整个基因组的异常。研究表明染色体区段拷贝数增加和减少即拷贝数变异(copy number variationCNV)直接造成位于该区段的基因拷贝数发生变化,影响基因表达水平,导致表型差异。抑癌基因所在的染色体区段的杂合性缺失(loss of heterzygosity LOH)是一些上皮和间质来源的肿瘤发生、发展过程中的重要事件。肺癌遗传变异的研究方法主要包括核型分析,荧光原位杂交(fluorescent in situ hybridizationFISH),比较基因组杂交(comparative genomic hybridization CGH)等。CGH只能检测到扩增或缺失数目的异常,但不能发现Allelic homozygosity(AH),这需要研究在肿瘤细胞中是一种非常常见的DNA变异一杂合性缺失,抑癌基因的杂合性缺失会导致肿瘤的发生,全基因组扫描LOH传统方法包括微卫星标记法(polymorphic microsatellite markers)和限制性片段长度多态性(retriction fragment length polymorphism,RFLP),但是这两种方法很费时费劲。SNP是人类基因组里最普遍的多态性遗传标志。Affymectrix Genome-wide SNP 6.0芯片选取约906,600个位点分析各位点的基因型,同时芯片又能提供各位点两种等位基因型的拷贝数(CN),得到位点所在区段的CN和LOH情况,对基因组分析达到极高的分辨率(<10kb)。本研究的目的是通过应用SNP芯片对肺癌肿瘤组织进行全基因组扫描,探讨肺癌组织基因拷贝数扩增、缺失,以及杂合性缺失等遗传变异与肺癌发生的相关性,旨在揭示肺鳞癌发生的分子机制,提高早期诊断和提供治疗新方法。本研究应用SNP芯片检测15例肺鳞癌全基因组CNV和LOH遗传变异信息,进一步研究全基因组的拷贝数扩增,缺失,以及杂合性缺失等染色体异常,从全基因组水平对肺鳞癌的分子遗传机制异常进行初步探讨。方法提取15例肺鳞癌患者的肺癌组织和配对的远癌肺组织的全基因组DNA并用于Affymetrix Genome-wide SNP 6.0芯片检测。芯片探针杂交信号强度的数据使用Affymetrix专用软件GTC3.0分析SNP位点基因型和拷贝数得到全基因组的CNV,LOH,纯合性缺失区域等信息。结果1.Genome-wide SNP 6.0芯片的QC Call rate都超过了质控标准。15张芯片的QC Call rate最低的为95.07%,最高的为99.26%,平均为97.49%,Contrast QC最低为2.08,最高为3.75,平均为2.73,MAPD最低为0.16,最高为0.28,平均为0.22,表明芯片杂交质量良好。2.15例肺癌组织分析结果中CN为2的片段共181个,占1.26%,片段长度最短的为100kb,最长的为3,995kb。CN为4的片段共226个,占1.57%,片段长度最短的为100kb,最长的为34,862kb。CN为3的片段共6,018个,占41.9%,片段长度最短的为100kb,最长的为25,654kb。CN为1的片段共7,837个,占54.6%,片段长度最短的为100kb,最长的为86,897kb。3.15例肺癌组织中CNV中Loss主要分布在第4号染色体(7.11%,1001/14081),3号染色体(6.22%,876/14081),5号染色体(4.92%,693/14081),1号染色体(4.06%,572/14081),10号染色体(3.14%,442/14081)等染色体上,gain主要分布在7号染色体(4.99%,702/14081),2号染色体(4.57%,643/14081),8号染色体(4.09%,576/14081),1号染色体(3.88%,547/14081),12号染色体(3.49%,491/14081)等染色体上。其中gain片段数量最少的是C08-0342,共7个片段,最多的是C08-0027,共877个片段；loss片段数量最少的是C08-0085,共4个片段,最多的是C08-0027,共1,628个片段。4.15例肺癌组织分析结果中都有不同程度的杂合性缺失,15例肺癌组织检测出各个样本含有不同数量的LOH位点。15例肺癌组织总共检出1,921,845个LOH位点,其中LOH位点数量最少的为C08-0142,共计54,274个LOH位点,LOH位点数量最多的为C08-0027,共计400,078个LOH位点。其中不同染色体也有不同的LOH位点数量,其发生率由高至低依次前5条染色体为第X号染色体(28.1%,540105/1921845),第1号染色体(7.14%,137282/1921845),第5号染色体(6.89%,132347/1921845),第3号染色体(6.19%,119014/1921845),第4号染色体(6.11%117336/1921845)。15例肺癌组织的X染色体上均能检测到LOH位点,共检测到31,793个LOH位点。5.15例肺癌组织分析结果中检测到纯合性缺失区域(CN=0)共92处,分别位于第1,2,3,4,5,6,7,8,9,10,12,13,14,15,16,17,18,19,21,X,Y号染色体上,其发生率由高到低依次为第4号染色体(21.7%,20/92),第5号染色体(11.9%,11/92),第3号染色体(9.8%,9/92),Y染色体(8.7%,8/92),第X染色体(7.6%,7/92),第19号染色体(6.5%,6/92),第17号染色体(5.4%,5/92),第1号染色体(4.3%,4/92),第8,10,15,18号染色体(3.3%,3/92),第7号染色体(2.2%,2/92),第2,6,9,12,13,14,16,21号染色体(1.1%,1/92)。最小的缺失片段在第19号染色体的q13.1,长度为101kb,最长的缺失片段在第3号染色体的p12.3～p12.2,长度为7,146kb。结论(1)Genome-wide SNP 6.0基因芯片和分析软件Genome Typing Console 3.0,可以同时详细分析肺鳞癌的染色体缺失,扩增和LOH,纯合性缺失区域；(2)Genome-wide SNP 6.0基因芯片筛选出多处肺鳞癌相关基因,表明多个基因可能参与了肺鳞癌的发生,发展过程；(3)发现的扩增和纯合性缺失以及杂合性缺失区域可能含有新的癌基因或抑癌基因,为今后的研究提供了新的线索和方向。更多还原

【Abstract】 Object In the last few years,reports have demonstrated that copy number gain/or loss (copy number variation CNV) influences gene expression and result in phenotypic variation by disrupting genes and altering gene dosage.Loss of heterozygosity (LOH) of chromosomal regions bearing tumor suppressor genes is a key event in the oncogenesis and development of epithelial and mesenchymal tumors. But the functions of LOH and CNV have not been fully understood. Previous studies attempting to identify genetic changes occurring in non small cell lung cancers have relied on cytogenetic studies, fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH).LOH can be analysed through allelotyping of tumors with polymorphic genetic markers.Restriction fragment length polymorphism (RFLP) or microsatellites are reliable genetic markers, but the genotyping procedure is rather laborious and time-comsuming on a genome-wide scale.Genome-wide detection of LOH, as well as CNV in cancer genomes, has drawn recent attention in the field of cancer genetics.SNPs are the most common form of sequence variation in the human genome, occurring approximately every～1000 bp.Affymetrix Single Nucleotide Polymorphism Microarray was originally developed for large-scale genome-wide association studies, provide a powerful platform for both genome-wide LOH analysis and CN detection. In this platform, the use of large numbers of SNP-specific probes showing linear hybridization kinetics allows not only high-resolution(<10Kb) LOH analysis at 906,600 SNP loci but also accurate determination of the CN status at each LOH region compared with the low resolution of array-CGH. The aim of this study is to obtain the genetic changes of non small cell squamous lung cancer by using Affymetrix genome-wide SNP 6.0 array for screening out genes that are related to their phenotypes, and ultimately try to study the melocular mechanisms of non small cell squamous lung cancer.Method Genomic DNA extracted from 15 non small cell squamous lung cancer tissues and matched normal lung tissues were used to be analysed by genome-wide SNP 6.0 arrays.The hybridization intensity of each probe was analyzed using Affymetrix proprietary software for genotyping and copy number at each locus and inorder to obtain global profiles of CNV and LOH of the tumors.Result 1.The SNP call rate of 15 arrays ranged from 95.07% to 99,26%,with average call rate of 97.49%. QC call rate of the 15 arrays ranged from 2.08 to 3.05,averaged 2.73.All the call rates of the arrays were in excess of 93%,the cardinal quality control standard.2.Of the 15 arrays,There are 7837 regions which CN=1,accounted for 54.6% of all the CNV regions; There are 6018 regions which CN=3,accounted for 41.9% of all the CNV regions;There are 226 regions which CN=4,accounted for 1.57% of all the CNV regions; The majority of the chromosome loss alterations were located on chromosome 4,3,5,1,10;The majority of the chromosome gain alterations were located on chromosome 7,2,8,1,12.3.LOH was detected in all the 15 arrays with different degrees,a total number of 1,921,845 were detected in the 15 arrays.The minimum number of 54,274 LOH was detected in sample C08-0142 and the maximum number of 400,078 LOH was detected in sample C08-0027.The percentage of LOH incidence rate ranged from 0.03% to 28.1%.4.92 homozyous deletion regions were detected in the 15 arrays,which were located on chromosome1,2,3,4,5,6,7,8,9,10,12,13,14,15,16,17,18,19,21,X,Y.The percentage of HD occurred among all the chromosome were chromosome 4(21.7%), chromosome 5(11.9%),chromosome 3(9.8%),chromosome Y(8.7%),chromosome X(7.6%),chromosome 19(6.5%),chromosome 17(5.4%),chromosome 1(4.3%), chromosome 8,10,15,18(3.3%),chromosome 7(2.2%),chromosome 2,6, 9,12,13,14,16,21(1.1%).The shortest HD region was located on q13.1 of chromosome 19,covering 101kb;the longest HD region was located on p12.3～p12.2 of chromosome 3,covering 7,146kb.Conclusion(1)Genome-wide chromosone copy number variation and LOH can be detected by using high resolution Affymetrix Genome-wide SNP 6.0 array simultaneousl.(2) We could identify narrow amplified and deleted region to find new oncogenes and tumor suppressor genes by using the Affymetrix Genome-wide SNP 6.0 array and software Genome Typing Console 3.0.(3).The novel amplified or deleted genes discovered here might beeome targets for the early diagnosis and therapy for non small cell squmous lung cancer.更多还原