【作者】 王新；

【导师】 樊代明； 金建平；

【作者基本信息】 第四军医大学 ， 内科学（消化系病）， 2001， 博士

【摘要】 肿瘤的多药耐药现象是肿瘤化疗的主要障碍。目前发现的肿瘤多药耐药机制主要涉及Pgp、MRP、LRP、BCRP、GSH/GST、PKC、TopoⅡ、DNA修复、多种凋亡相关基因和肿瘤细胞生活的内外环境（如pH、缺氧、温度）变化。但有关胃癌MDR的机制还所知甚少，初步的研究显示胃癌MDR有其特殊性，现有的MDR机制还不能圆满解释和逆转胃癌的多药耐药性。 目的 建立和优化蛋白质组学分析中的双向聚丙烯酰胺凝胶电泳技术，寻找胃癌细胞耐药相关蛋白质；分离、克隆胃癌细胞中耐药相关的差异表达基因，阐明胃癌细胞耐药新的分子机制。 方法 建立和优化蛋白质组学分析中的双向聚丙烯酰胺凝胶电泳技术 （O’Farrell系统和IPG-等电聚焦双向电泳系统），并用其比较胃癌SGC7901细胞和长春新硷耐药细胞SGC7901/VCR蛋白质组的差异，凝胶银染法显示其蛋白质的差异表达，进而用质谱测序鉴定这些差异表达蛋白；应用改进的mRNA差异显示技术分析这两种细胞中显著差异表达的cDNA，经反向Northern分析、差异基因序列测定和Northern Blot分析确证差异表达基因。进一步用RACE和MTE Array分析差异表达基因。 结果 建立并优化了蛋白组学分析中的双向电泳技术—O’Farrell双向电泳系统和IPG-等电聚焦双向电泳系统，应用此双向电泳技术比较胃癌SGC7901细胞和其长春新硷耐药亚系SGC7901/VCR细胞蛋白质组的差异。结果发现30种明显差异表达的蛋白质，并初步确定了其等电点和分子量。其中3个蛋白点表达量很高但 第四军医大学博士学位论文 一 在非耐药细胞图谱中未出现；6个蛋白点丰度明显上调；19个蛋白点丰度明显下调； 2个蛋白点在耐药细胞图谱中未出现但在非耐药细胞中高表达。把差异蛋白转印到 PVDF ＄上，对其中一个差异蛋白质进行了测序鉴定分析，同源性检索显示为一未 知的蛋白质。应用mRNA差异异示技术从胃癌SGC7901和SGC7901／VCR细胞中筛选 并鉴定了20个在SGC7901／VCR细胞中特异或高表达的基因，其中7个基因片段与 己知基因高度同源，部分己知基因与耐药相关。另外13个CDNA片段为未知基因， 对其中的毛个cDNA片段进行了Northern分析，结果再次表明它们在VCR细胞中特 异或高表达。它们的 mRNA全长分别为 GRP－2。1．gkb：GRP－28，1、6kb：GRP19，2．okb： GRP32，1．7kb。对 GRP－2片段用 3 RACE法扩增其全长达 1．Zkb，仍为一新的基因。 MTE Arraa杂交分析显示 GRP－2主要在正常肺组织内表达，其它组织细胞中弱表达。 GRP－2很可能是在胃癌VCR耐药机制中很重要的基因。 结论 在胃癌SGC7901／VCR发现了30种明显差异的蛋白质，其中3种蛋白质在VCR 耐药细胞中特异高表达。同时发现20种基因在胃癌SGC7901／VCR细胞中高表达，其 中 7个基因片段与已知基因高度同源，部分已知与耐药相关。13个 CDNA片段为未知 基因，其中GRP－2在耐药细胞中特异高表达。这些差异表达的蛋白质和基因分子可 能与胃癌细胞长春新碱耐药机制相关，为进一步研究胃癌MDR机制奠定了物质基础。更多还原

【Abstract】 Multi-drug resistance (MDR) of tumor cells is the main obstacle of cancer chemotherapy. Previous studies have shown that the mechanisms of MDR involve Pgp, MRP, LRP, BCRP, GSH/GST, PKC, Topo DNA plerosis, genes related to apoptosis and changes of cellular environment (such as pH, hypoxia and temperature). Very little is known for MDR of gastric carcinoma cells. Preliminary studies have shown specific features in MDR of gastric carcinoma cells, which cannot be completely explained by any known mechanisms of MDR.Aim of the study: This research project aimed to investigate the expression and regulation of MDR-related proteins and genes in gastric cancer SGC7901 cells and Vincristine-resistant SGC7901 (SGC7901/VCR) cells for significance in the development of MDR in cancer cells.Methods: Two-dimensional (2-D) polyacrylamide gel electrophoresis (PAGE) using O’Farrell system and immobilized pH gradients (IPG) were applied to compare the differential expression of MDR proteins in gastric cancer SGC7901 and SGC7901/VCRcells. The differentially expressed proteins were identified by matrix assisted laser desorption ionization mass spectrometry (MALDI-MS). A modified differential-display polymerase chain reaction (DD-PCR) was used to examine the expression of mRNA in SGC7901/VCR cells versus SGC7901 controls. The differentially expressed mRNA were cloned and the cDNA fragments were confirmed by reverse-Northern blot hybridization, sequencing analysis and Northern bolt analysis. One of the cDNAs was further analyzed by3’-RACE and multiple tissue expression (MTE) array.Results: This study has optimized the 2-D PAGE methods to demonstrate the differentially expressed proteins in SGC7901 and SGC7901/VCR gastric cancer cells. Thirty proteins were found significantly different in their expression levels with 6 higher and 19 lower in SGC7901/VCR cells. Five proteins were found to be unique to SGC7901 or SGC7901/VCR cell (3 in SGC7901/VCR cells, 2 in 7901 cells). One of the differentially expressed proteins was sequenced by MALDI-MS. Identified by DD-PCR, 20 cDNAs were found with higher expression in SGC7901/VCR cells. Sequencing analysis revealed that seven of the mRNAs were encoded by known genes which may be related to MDR. Thirteen of the cDNA were products of unknown genes. The size and abundance of 4 of the novel gene products were confirmed by Northern blot analysis (GRP-2, 1.9kb; GRP-19, 2.0kb; GRP-28, 1.6kb; GRP-32, 1.7kb, respectively). Results confirmed their high expression level in SGC7901/VCR cells. GRP-2 was further characterized by MTE array hybridization to show its high expression in normal lung and weak expression in other histocytes, suggesting that it may be an important gene in MDR of gastric carcinoma.Summary: Novel proteins with regulated expression were found in gastric cancer SGC7901/VCR cell line. Twenty cDNAs were found with up-regulated expression ingastric cancer SGC7901/VCR cells. Cloning and sequencing revealed that seven of them encode known proteins which may be related to MDR. Thirteen of the cDNAs are products of unknown genes with GRP-2 showing up-regulated expression in SGC7901/VCR cell. These differentially expressed proteins and genes provide materials for further investigation on the molecular mechanism of MDR.更多还原