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目的：建立优化的差示PCR条件；运用建立的条件分离胃癌GC7901与胃粘膜GES－1细胞株之间的差异表达基因；根据获取的序列，设计引物并运用于检测胃癌组织、血、活检胃粘膜标本，拟筛选出检出率与符合率高的数对引物，建立胃癌基因诊断方法．方法：LJGC7901与GES－1细胞株为研究对象，探索mRNA差示PCR的最佳反应条件，运用优化的条件分离细胞株之间的差异表达基因；以回收的片段作探针，打点杂交证实为真正的差异片段后，对产物进行直接测序及同源性检索；设计引物，采用RT－PCR方法检测胃癌组织、血、活检胃粘膜标本．结果：优化的差示PCR条件为：前5轮PCR循环采用94℃35s，40℃2min，72℃30s，后35轮PCR循环采用94℃45s，55℃2min，72℃1min，最后在72℃延伸7min；分离回收差异条带154条，从中选出17个片段作探针，打点杂交证实在两细胞株之间呈差异表达；对17个片段进行了测序，其中17个新序列被GenBank接受，接受号为AF054162至AF054172，AF071052至AF071058；其中5个序列引物在26例胃癌标本中检出覆盖率为100％，在9例病理确诊的胃癌患者活检胃粘膜中检出率100％，在17例病理确诊为胃癌患者血标本中检出率为53％．结论：优化了mRNA差示PCR技术条件；分离出154个差异表达的基因片段；筛选出17个新序?
Objective: To establish optimized conditions for differential PCR; to use established conditions to isolate differentially expressed genes between gastric cancer GC7901 and gastric mucosal GES-1 cell lines; based on the sequence obtained, primers were designed and applied to the detection of gastric cancer, blood, and biopsy stomach Mucosal specimens, to be selected by the number of pairs of primers with high detection rate and coincidence rate, to establish genetic diagnosis of gastric cancer. METHODS: LJGC7901 and GES-1 cell lines were investigated. The optimal reaction conditions for mRNA differential PCR were explored. Differentially expressed genes were separated between the cell lines using optimized conditions. The recovered fragments were used as probes and dot hybridization was verified as After the actual differential fragments, the products were directly sequenced and homologously searched; primers were designed and RT-PCR was used to detect gastric cancer tissues, blood, and biopsy gastric mucosa specimens. RESULTS: The optimized differential PCR conditions were: first 5 cycles of PCR using 94°C 35s, 40°C 2min, 72°C for 30s, followed by 35 cycles of PCR using 94°C 45s, 55°C 2min, 72°C 1min, and finally 72°C The extension was 7min; 154 differential bands were isolated and recovered, and 17 fragments were selected as probes. Dot blot hybridization confirmed differential expression between the two cell lines; 17 fragments were sequenced, and 17 new sequences were accepted by GenBank. The acceptance numbers were AF054162 to AF054172, AF071052 to AF071058; of these, 5 primers detected the coverage rate of 100% in 26 specimens of gastric cancer, and the detection rate was 100% in the biopsy gastric mucosa of 9 patients with pathologically confirmed gastric cancer. The detection rate of 17 cases of pathologically confirmed gastric cancer patients was 53%. Conclusion: Optimized mRNA differential PCR technology conditions; Isolated 154 differentially expressed gene fragments; Screened 17 new sequences?