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目的 筛选胃癌下调基因 ,确定其表达产物的组织分布和细胞定位。方法 从已成功建立的 5人份正常胃粘膜mRNA(Tester)抑制消减杂交胃癌mRNA(Driver)的差异表达文库 ,随机挑取阳性克隆测序为CA11,RT -PCR证实其为下调基因。地高辛标记CA11原位mRNA杂交。在其它组织器官中扩增此基因 ,以表明其组织分布特异性。CA11克隆至载体 pcDNA3.1/Myc -His(- )A中 ,并瞬时转染CA11的COS - 7细胞。生长至对数生长期时 ,吸取培养液 ,Talon吸附、洗脱后 ,取洗脱液进行SDS蛋白电泳及Westernblot。CA11转染COS - 7细胞后加抗Myc标签的一抗 ,最后加FITC标记的二抗在荧光显微镜下观察。结果 筛选到胃癌下调基因CA11,并经RT -PCR证实。mRNA原位杂交显示 ,CA11位于胃粘膜上皮细胞。在其它组织器官cDNA文库及组织RNA中均未扩增出该基因。洗脱液Westernblot未检测到CA11与pcDNA3.1/Myc -His(- )A的Myc融合表达蛋白存在 ;CA11表达蛋白亚细胞定位于细胞浆。结论CA11为一胃癌下调基因 ,它在胃粘膜上皮细胞特异表达 ,表达产物定位于细胞浆
Objective To screen down-regulated genes in gastric cancer and determine the tissue distribution and cellular localization of the expressed products. METHODS: Differential expression library of hybrid gastric cancer mRNA (Driver) was successfully established from 5 normal gastric mucosa mRNA (Tester). The positive clones were randomly selected and sequenced as CA11. RT-PCR confirmed that they were down-regulated genes. Digoxigenin labeled CA11 in situ mRNA hybridization. This gene is amplified in other tissues and organs to show its tissue-specific distribution. CA11 was cloned into vector pcDNA3.1 / Myc-His (-) A and transiently transfected into COS-7 cells of CA11. Growth to logarithmic growth phase, the culture medium was taken, Talon adsorption, after elution, take the eluent for SDS protein electrophoresis and Westernblot. CA11 was transfected into COS - 7 cells and then labeled with anti - Myc antibody. Finally, FITC labeled secondary antibodies were observed under a fluorescence microscope. Results The gastric cancer gene CA11 was screened and confirmed by RT-PCR. In situ hybridization showed that CA11 was located in gastric mucosal epithelial cells. This gene was not amplified in other tissue and organ cDNA libraries and in tissue RNA. Western blot analysis showed that there was no detectable expression of Myc fusion protein between CA11 and pcDNA3.1 / Myc-His (-) A, and CA11 protein subcellular localization in cytoplasm. Conclusion CA11 is a gastric cancer down-regulated gene, which is expressed specifically in epithelial cells of gastric mucosa and localized in cytoplasm