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为了分离鼻咽癌差异表达基因 ,应用抑制性扣除杂交技术 ,在正向抑制性扣除杂交中 ,以鼻咽癌上皮细胞株HNE1cDNA作为检测子 ,以人胚鼻咽上皮细胞cDNA作为驱赶子 ;在反向抑制性扣除杂交中 ,以人胚鼻咽上皮细胞cDNA作为检测子 ,以鼻咽癌上皮细胞株HNE1cDNA作为驱赶子 ,分别通过抑制性扣除杂交 ,构建了鼻咽癌上皮细胞株HNE1表达下调和表达上调的两个扣除cDNA文库 .从鼻咽癌相关的扣除cDNA文库中随机挑取 1 2 0 0个克隆 ,采用菌落PCR扩增其插入cDNA片段 ,自动点膜制备成cDNA微阵列膜 ,分别用鼻咽癌上皮细胞株HNE1、人胚鼻咽上皮mRNA经逆转录标记cDNA探针 ,分别与cDNA微阵列膜杂交 ,通过杂交信号的自动扫描分析 ,对杂交信号存在 5倍差异的克隆进行测序 ,获得了 1 0个鼻咽癌差异表达基因的cDNA片段 ,其中 3个为新基因序列 ,其GenBank登录号为 :AF5 1 0 1 88、AF5 1 0 1 89和AF5 1 0 1 90 ,7个代表已知基因序列 .采用RT PCR证实S1 0 0A8,CK1 9和RBP1基因在人胚鼻咽上皮中高表达而在鼻咽癌细胞株HNE1中低表达 .这些结果显示上述基因可能是鼻咽癌发生的重要因素
In order to isolate the differentially expressed genes of nasopharyngeal carcinoma, suppressive subtractive hybridization (SSH) was used to detect the nasopharyngeal epithelial cell line HNE1cDNA as detector and the cDNA of human embryonic nasopharyngeal epithelial Reverse repression subtractive hybridization using human embryonic nasopharyngeal epithelial cell cDNA as a detector to NPC nasopharyngeal epithelial cell line HNE1cDNA as a driver, respectively, by inhibition subtractive hybridization, the nasopharyngeal epithelial cell line HNE1 expression was down-regulated And two up-regulated cDNA subtracted cDNA library were randomly selected from subtracted cDNA library related to nasopharyngeal carcinoma and cloned into cDNA fragments by colony PCR, Nasopharyngeal epithelial cell line HNE1 and human embryonic nasopharyngeal epithelial mRNA were respectively reversely labeled with cDNA probe and hybridized with cDNA microarray respectively. The hybridization signal was analyzed by auto scanning, and clones with 5-fold difference in hybridization signal The cDNA fragments of 10 differentially expressed genes in nasopharyngeal carcinoma were obtained, of which 3 were novel gene sequences and their GenBank accession numbers were AF5 1 0 1 88, AF5 1 0 1 89 and AF5 1 0 1 90 , 7 representatives of known gene sequence.RT-PCR confirmed that S100A8, CK1 9 and RBP1 genes were highly expressed in human embryonic nasopharyngeal epithelium but low in nasopharyngeal carcinoma cell line HNE1.These results suggest that these genes may be nasal An important factor in the occurrence of pharyngeal cancer