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目的建立优化的ｍＲＮＡ差示ＰＣＲ条件；运用建立的条件及ＧＱＳ９６００分离胃癌ＧＣ７９０１与胃粘膜ＧＥＳ１细胞株之间的差异表达基因片段；根据获取的序列，设计引物并运用于胃癌组织、血、活检胃粘膜标本检测，拟从中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．方法以胃癌ＧＣ７９０１与胃粘膜ＧＥＳ１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片段；以回收的片段作探针，打点杂交证实为真正的差异片段后，对纯化产物进行直接序列分析及ＧｅｎＢａｎｋ同源性检索，同源性低的序列递交给ＧｅｎＢａｎｋ；设计引物，采用ＲＴＰＣＲ方法对胃癌组织、血、活检胃粘膜进行检测，筛选出检出率与符合率高的５对引物建立多重ＰＣＲ诊断胃癌方法．结果①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；②确认并分离出差异条带１５４条，胃癌细胞株泳道中存在而胃粘膜细胞株泳道中缺失的有８２条，胃粘膜细胞株泳道中存在而胃癌细胞株泳道中缺失的有３５条，胃癌细胞株泳?
Objective To establish an optimized mRNA differential PCR condition; to use the established conditions and GQS 9600 to isolate differentially expressed genes between gastric cancer GC7901 and gastric mucosa GES-1 cell lines; according to the sequence obtained, primers were designed and applied to gastric cancer tissues, Blood and biopsy specimens of gastric mucosa were tested and several pairs of primers with high detection rate and coincidence rate were selected to establish a genetic diagnosis method for gastric cancer. Methods Gastric cancer GC7901 and gastric mucosal GES1 cell line were investigated to explore the optimal reaction conditions of mRNA differential display PCR. Optimum conditions were used to isolate differentially expressed gene fragments between cell lines. The recovered fragments were used as probes. After the hybridization was confirmed as a true differential fragment, the purified product was subjected to direct sequence analysis and GenBank homology search. The sequence with low homology was submitted to GenBank; primers were designed and RT-PCR was used to analyze gastric cancer tissue, blood, and biopsy stomach. The mucous membranes were detected and 5 pairs of primers with high detection rate and coincidence rate were screened to establish multiplex PCR for diagnosis of gastric cancer. RESULTS 1 Optimized differential PCR conditions: first 5 cycles of PCR using 94°C 35s, 40°C 2min, 72°C 30s, 35 cycles of PCR using 94°C 45s, 55°C 2min, 72°C 1min, and finally 72°C Extend 7 min; 2 Confirm and isolate 154 differential bands, which are present in the lanes of gastric cancer cell lines and 82 in the lanes of gastric mucosal cell lines, which are present in the lanes of gastric mucosal cell lines and absent in the lanes of gastric cancer cell lines. Article, gastric cancer cell strain?