【作者】 陈照立；

【导师】 李君文；

【作者基本信息】 中国人民解放军军事医学科学院 ， 劳动卫生与环境卫生学， 2007， 博士

【摘要】 研究目的：目前已知的与二羟环氧苯并芘(BPDE)相互作用的基因尚不能阐明BPDE的致癌分子机制，寻找出更多的BPDE致癌易感基因是个亟需解决的问题。本研究的目的是建立一种新的方法技术，在小鼠全基因组水平筛选出更多的BPDE致癌易感基因，并初步探讨筛选基因在A549肺癌细胞中的作用，为研究BPDE的致癌分子机制打下基础。研究方法：本研究首次应用扩增片段长度多态性(amplified fragment length polymorphism，AFLP)与免疫磁性分离(immunomagnetic separation，IMS)技术相结合的方法，对小鼠全基因组DNA与BPDE相互作用的基因位点进行筛选，对差异片段进行了回收、再扩增、克隆测序及BLAST同源相似性检索分析，获得与差异核苷酸序列同源的已知基因或未知序列，并用RNA干扰(RNA interference，RNAi)技术对筛选出的EFNB2基因在A549肺癌细胞中的作用进行初步探讨。研究结果：1、以AFLP和免疫磁性分离技术相结合，成功建立了基于小鼠全基因组的高效、特异筛选BPDE致癌相关基因的新方法。2、成功获得了清晰的变性聚丙烯酰胺凝胶电泳条带图谱和与BPDE作用的差异DNA片段条带图谱，并成功对差异片段进行了回收、扩增、克隆测序，共获得125条差异片段DNA的核苷酸序列。3、经BLAST分析发现：有193条已知基因与差异片段核苷酸序列有同源性，主要为信号转导及通路、生长发育、血管生成、免疫及炎性反应、转录调控、细胞分化等方面的基因和一些未知功能的基因。有6条未知核苷酸序列，向GenBank提交注册，已被GenBank接受为新序列并给予相应的基因登录号：分别为EF176681、EF473140、EF473141、EF473142、EF473143、EF473144。这些基因和序列有可能是与BPDE致癌相关的基因序列。4、经RT-PCR和免疫荧光染色证实，A549肺癌细胞中EFNB2基因mRNA和蛋白均有较高的表达水平。5、EFNB2基因RNAi质粒表达载体转染A549肺癌细胞后，可以抑制A549肺癌细胞中EFNB2基因mRNA和蛋白的表达。6、平板克隆形成实验中，以A549-mocks组形成的克隆个数最高，为45.8±3.27个，其次是A549-pGPU6/GFP/Neo-shNC阴性对照为43.6±3.21个，A549-pGPU6/GFP/Neo-shEFNB2/159克隆形成数最低(37.6±5.64个)，shEFNB2/159组形成的克隆数与其他两组相比差异有统计学意义，说明RNAi对EFNB2基因表达的抑制可能对A549肺癌细胞的体外增殖能力产生了抑制作用。7、在与血管内皮细胞的粘附实验中，A549-mocks组的A549细胞的A值平均为0.151，shNC为0.141，shEFNB2/159组的A值最低，平均为0.081，shEFNB2/159组与两个对照组相比较差异均有统计学意义，提示EFNB2基因沉默可能抑制了A549肺癌细胞对血管内皮细胞的体外粘附能力。8、Transwell侵袭小室实验中，侵袭穿过微孔膜的A549细胞数在mocks对照组(59.80±5.76个)和shNC阴性对照组(54.80±4.87个)较多，在shEFNB2/159组穿膜细胞数明显减少(25.20±3.90个)，shEFNB2/159组穿膜细胞数与两个对照组相比较差异有统计学意义，结果表明，EFNB2基因沉默可能抑制了A549肺癌细胞的体外侵袭能力。研究结论：1、扩增片段长度多态性(AFLP)和免疫磁性分离(IMS)技术相结合的方法，适用于在小鼠全基因组筛选BPDE致癌相关基因。2、应用AFLP和IMS相结合的方法，共筛选出与差异片段核苷酸序列有同源性的已知基因193条；筛选出未知核苷酸序列6条，已被GenBank接受为新序列并给予相应的基因登录号：EF176681、EF473140、EF473141、EF473142、EF473143、EF473144。这些基因和序列有可能是与BPDE致癌相关的基因序列。3、通过RNA干扰技术沉默EFNB2基因表达，可以显著抑制A549肺癌细胞的体外增殖、体外粘附和体外侵袭能力。更多还原

【Abstract】 Objective The carcinogenesis mechanism of benzo[α]pyrene dihydrodiol epoxide (BPDE) is still unclear according to the known genes interaction with BPDE. So it is an urgent problem to screen more BPDE target susceptible genes. The purposes of this study were to found a new method to screen more BPDE target susceptible genes from whole genomes in mice, to make a preliminary study using RNAi to explore the role of EFNB2 gene silencing in A549 lung cancer cell lines, and to found a basis for study on carcinogenesis mechanism of BPDE.Methods Amplified fragment length polymorphism (AFLP) combination with immunomagnetic separation (IMS) were first used to screen, enrich and amplify the fragments of BPDE interaction with DNA in mice whole genomes. Differential fragments were displayed by polyacrylamide gel electrophoresis and Silver nitrate staining. The Differential fragments were collected, cloned, sequenced, and compared homology similarity with the known genes in GenBank BLAST. RNA interference (RNAi) was used to explore the role of EFNB2 gene silencing in A549 lung cancer cell lines.Results(1) A new effective and specific method, AFLP combination with IMS, was first successfully founded to screen BPDE target susceptible genes from whole genomes in mice.(2) Distinct electrophoresis patterns were obtained by denatured polyacrylamide gel electrophoresis (DPAGE) and differential bands of DNA fragments interaction with BPDE were displayed in DPAGE electrophoresis pattern. The differential bands DNA were successfully collected, cloned, sequenced, and 125 nucleotide sequences were obtained.(3) Homology similarity was compared.with the known genes in GenBank BLAST. 193 known genes sequences in GenBank mouse genomes database showed homology similarity with the differential bands DNA sequences, the function of these 193 genes is in different categories, such as signal transduction, development, angiogenesis, immune and inflammatory response, regulation of transcription, cell differentiation, etc; The function of some genes among 193 genes is still unknown today. 6 sequences showed no significant similarity in GenBank mouse genomes database, These 6 sequences were submitted to GenBank database, which assigned accession numbers are EF176681, EF473140, EF473141, EF473142, EF473143, EF473144, respectively. These genes and sequences may be BPDE-induced tumor associated genes or sequences.(4) EFNB2 mRNA and protein expressed abundantly in A549 lung cancer cell lines according to the results of RT-PCR and immunofluorescence.(5) Small interference RNA (siRNA) expressed by RNAi plasmid expression vector can inhibit the expression of EFNB2 mRNA and protein in A549 lung cancer cell lines.(6) The number of A549 lung cancer cells clone formation, A549 lung cancer cells adhesion to vascular endothelial cells, A549 lung cancer cells invasion through Matrigel micropore membrane in vitro significantly decreased when EFNB2 gene silencing compared with control groups. Silencing of EFNB2 gene expression may significantly inhibit A549 lung cancer cells’ proliferation, adhesion and invasion ability in vitro.ConclusionsOur data suggest that AFLP combination with IMS as a new effective and specific method can be used to screen BPDE target susceptible genes from whole genomes in mice, that the genes and sequences abtained by AFLP combination with IMS may be BPDE-induced tumor associated genes or sequences, and that EFNB2 gene silencing may significantly inhibit A549 lung cancer cells’ proliferation, adhesion and invasion ability in vitro.更多还原