摘要
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 .
In order to isolate and identify differentially expressed genes in nasopharyngeal carcinoma (NPC), two processes of SSH were performed. In the forward subtraction, cDNAs from primary cultural human embryo nasopharyngeal epithelium were used as tester and NPC cell line HNE1 as driver. In the reverse subtraction, primary cultural human embryo nasopharyngeal epithelium were used as driver and NPC cell line HNE1 as tester. Two NPC cell line HNE1 related subtractive cDNA libraries were constructed following the above two SSH processes. 1200 colonies were randomly picked up and their inserts were amplified by PCR, and arrayed onto nylon membranes using robotic printing. The differential gene expression was then verified by the hybridization of the membrane with radioactively labeled first strand cDNAs prepared from NPC cell line HNE1 and primary cultural human embryo nasopharyngeal epithelium. Five fold differentially expressed clones were picked up in accordance with their hybridization signal intensities for further analysis, including direct sequencing. Ten differentially expressed genes including 3 novel fragments with GenBank accession numbers AF510188,AF510189 and AF510190 were obtained. Among these genes, it was identified that S100A8, CK19 and RBP1 genes were down expressed in NPC cell line HNE1. The results indicate that these genes may be important for nasopharyngeal tumorigenesis.
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
2003年第6期743-750,共8页
Chinese Journal of Biochemistry and Molecular Biology
基金
国家 8 63项目 (No .2 0 0 1AA2 2 10 3 1)
973重点项目 (No .G19980 5 10 0 8)资助~~
关键词
鼻咽癌细胞株HNEl
抑制性扣除杂交
CDNA微阵列
差异表达基因
nasopharyngeal carcinoma cell line HNE1, suppression subtractive hybridization, cDNA microarray, differentially expressed genes
