【作者】 金晓航；

【导师】 樊代明；

【作者基本信息】 中国人民解放军第四军医大学 ， 内科学， 2003， 博士

【摘要】 胃癌是我国发病率最高的肿瘤，而多药耐药是肿瘤化疗中的主要障碍。虽然在其它肿瘤多药耐药研究中已经发现了多种耐药相关分子，但是只有部分在胃癌中表达，或者只能部分逆转胃癌细胞耐药。所以就产生了一个问题，胃癌是否有特异性耐药相关分子?本研究所以胃癌细胞系SGC7901为亲本细胞，采用剂量递加的方法，建立了几种胃癌多药耐药细胞系。采用削减杂交和DD-RT-PCR技术，从胃癌多药耐药细胞系SGC7901／VCR与其亲本细胞中差示出一批胃癌多药耐药细胞中上调或者下调的基因。本文以在胃癌多药耐药细胞系SGC7901／VCR中上调基因片断V62为研究对象，通过转基因方法检测其与胃癌多药耐药表型的关系，鉴定其是否为胃癌多药耐药相关基因。 目的：研究差示基因V62与胃癌MDR的关系 方法： 1、BLAST同源分析在基因库寻找V62基因片断的同源基因； 2、RT-PCR扩增V62基因CDS全长； 3、利用pUCm-T载体克隆V62基因； 4、V62基因在胃癌多药耐药细胞系SGC7901／VCR与其亲本细胞中的Northern blot； 5、利用真核表达载体pcDNA3.1(+)构建V62基因的正、反义真核表达载体； 6、V62基因的体外转录与翻译； 7、电穿孔法转染细胞系建立； 8、转染细胞的MTT试验； 9、转染细胞的药物蓄积与潴留试验； 10、V62基因结构和功能的生物信息学分析。 结果： 1、以消减杂交所得到的在胃癌耐药细胞系SGC7901／VCR与其亲本细胞中差异表达的400bp左右的V62基因片段序列检索基因库，发现该基因片段与多种基因高度同源，通过相应的基因序列可以分为4类。这些基因都是假想蛋白，序列相似，构成一个基因家族。V62基因片断仅是这些基因C端的一部分，以其不足以推断究竟是那一个基因。 2、通过RT-PCR技术克隆该基因编码区全长。以OligodT为引物，以胃癌多药耐药细胞SGC7901／VCR的mRNA为模板，在逆转录酶AMV催化下逆转录反应90min，获得胃癌多药耐药细胞SGC7901／VCR中V62基因的cDNA；按照V62基因片段同源基因起始码和终止码位点保守序列分别设计上下游引物，以所获 第四军医大学博士研究生学位论文一CDNA为模板，在高保真 DNA聚合酶 Tq酶催化下扩增目的基因，分别获得900hp 和400hp 左右的基因。 3、通过蓝白筛选，将目的基因分别克隆入克隆载体pUCm—T；酶切反应证实，从克隆载体多克隆位点中均可以得目的基因大小的释放片断；测序，发现目的基因与假想蛋白 AF24和 AF编码区序列完全一致。 4、以目的基因为模板制备32P标随机引物，以看家基因fi一actin为内参照，与胃癌多药耐药细胞SGC7901／VCR及其亲本细胞InRNA进行N。ullem hi。t双杂交，证实V62基因在耐药细胞中上调，但是目的基因InRNA为2．4kb，与其所有同源基因皆不相同，提示其可能为人胃癌中的组织特异性基因转录产物。 5、在克隆载体基础上通过基因重组构建其正、反义真核表达载体 sV62—pcDNA3＊（十）和 asV62—pcDNA3＊（十＊构建的载体通过双酶切反应证实。 6、利用兔血红细胞体外转录和翻译系统进行基因表达，获得一个42KD的蛋白质，说明目的基因为蛋白质编码基因。 7、将正、反义真核表达载体载体通过电穿孔法分别转导胃癌多药耐药细胞SGC7901／VCR及其亲本细胞，用G418筛选，克隆化，分别建立稳定转染细胞系。转染正义真核表达载体的药敏细胞与转染空载体对照细胞相比，RTPCR方法检测到V62基因InRNA表达升高；转染反义真核表达载体的耐药细胞与转染空载体对照细胞相比，RTPCR方法检测到V62基因InRNA表达降低。 8、用MTT试验检测V62基因对胃癌多药耐药的影响，转染正义真核表达载体的药敏细胞对化疗药物的抗性增加，耐药性增强：而转染反义真核表达载体的耐药细胞对化疗药物的抗性减小，耐药性减弱。 9、通过药物蓄积和储留试验发现，转染正义真核表达载体的药敏细胞对化疗药物ADM的蓄积和储留减少；转染反义真核表达载体的耐药细胞对化疗药物ADM的蓄积和储留增多。以上结果提示，V62基因是胃癌多药耐药相关基因。 10、生物信息学分析，V62基因染色体定位于16q13；基因结构包含百个外显子，7个内含子；该基因在N端包含一个转甲基酶功能域，在C端包含一个DNA结合域。 结论：发现了一个新的胃癌MDR相关分子，InRNA为2．4kb，编码 31 ZAA，蛋白质分子量为 42KD，具有 DNA转甲基酶的主要结构特征，可能是一个胃癌MDR相关转甲基酶更多还原

【Abstract】 Stomach cancer is the most popular cancer in China with the highest mortality and multidurg resistance (MDR) is the main obstacle in chemotherapy. There have been many MDR related molecules found in the study of MDR, while only some of them are expressed in stomach cancer or they could only reverse the MDR in gastric cancer partly. So there is a question: is there any special MDR related molecule for stomach cancer? There are several MDR cell lines built in the institute of the PLA digestive diseases by laddering dose method with the gastric cancer cell line SGC7901 as the parental cell line. Furthermore, some upregulated or downregulated genes were displayed by subtractive hybridization or difference display reverse transcribe polymerase chain reaction(RT-PCR) between the MDR cell line SGC7901/VCR and its parental cell line. In this study we want to kown the relation between V62, one of the upregulated gene in MDR cell line SGC7901/VCR, and gastric cancer MDR and to identify if it’s a gastric cancer MDR related molecule by gene transfection.Objection: the relation between upregulated gene V62 and stomach cancer MDRMethods:1. Searching for the homologues gene of V62 in genebank by gene blast.2. Emply the CDS of V62 gene by RT-PCR3. Cloning V62 gene into cloning vector pUCm-T4. Displaying V62 gene between MDR cell line SGC7901/VCR and its parental cell line by Northern blot5. Construction of sense/antisense eukaryotic expression vector of V62 gene with eukaryotic expression vector pcDNA3.1(+)6. Transcribing and translating V62 gene in vitro7. Construction of gene transfection cell lines by electroporation8. Checking the drug sensitivity of gene transfection cell lines by MTT assay9. Checking the drug accumulation and retention in gene transfection cell lines by flow cytometer10. Prediction of the stricture and function of V62 gene by bioinformatics Results:1. Blasting in the genebank with the 300bp v62 gene difference displayed between MDR cell line SGC7901/VCR and its parental cell line, we found that there were several genes showing high homology with it. These genes were classified into 4 type according to their gene squenceso They were allhypothetical protein having the same sequences to form a gene family. Because the V62 gene snippet from subtractive hybridization as long as 300bp was too short to identified which gene it answering for.2. Cloned the CDS of V62 gene by RT-PCR. Taking OligodT as the primer, mRNA of SGC7901 /VCR cells as the templet, catalyzed by AMV for 2h, the cDNA of V62 gene was reverse transcribed; with 5’ and 3’ primers designed according to the starting site and ending site of CDS of V62 gene, and templet from V62 gene, a 900bp and a 400bp PCR products were gotten catalyzed by high fidelity DNA polymerase Taq polymerase3. Screened by blue-white method, the targeted gene were cloned into cloning vector pUCm-T separately; targeted gene snippets could be released by double endoenzyme digesting; sequencing results showed that the targeted gene have the completely same sequences to gene AF24 and AF11 encoding hypothetical protein separately4. V62 gene was higher expressed in SGC7901/VCR cells than its parental cells according to the double primer northern blot, in which the first probe was templeted by targeted gene and the second probe was templeted by home-keeper gene β-actin as the control. But the result showed that the mRNA of targeted gene was as long as 2.4kb, which is different to any reported gene in this family, so it suggested that this was a special gene transcribing product5. Eukaryotic expression vectors of V62 gene, sV62-pcDNA3.1 (+) # asV62-pcDNA3.1 (+) , were constructed on the base ofthe cloning vector and confirmed by double digestive reaction6. V62-939 gene was transcribed and translated into a 42KD protein in the rabbit homocell transcribing and translating system in vitro, which supported that V62 was a gene that could encoding a protein7. The sense/anti更多还原