NIH3T3细胞恶变早期相关新基因的鉴定与功能分析  

Identification and Functional Assay of Two Novel Genes in Early Stages of Transformed NIH3T3 Cells

作　　者：杨劲松[1] 郑新民[2] 陈诗书[1] 

机构地区：[1]上海第二医科大学生物化学教研室,分子生物学实验室,人类基因治疗研究中心,上海200025 [2]美国康奈尔大学分子生物学和遗传学系,纽约14850

出　　处：《中国生物化学与分子生物学报》2003年第2期173-181,共9页Chinese Journal of Biochemistry and Molecular Biology

摘　　要：利用已建立的蛋白质酪氨酸磷酸酶α(PTPα)诱导表达模型寻找与PTPα高表达导致NIH3T3细胞恶变早期相关的新基因 ,探索肿瘤早期形成的机理 .诱导PTPα表达 2 4h ,用差异显示逆转录PCR获得 65条差异片段 ,利用生物信息学方法分析这些差异片段 .发现其中含有 2 9种已知基因 ,12种已知ESTs ,6种未知ESTs .对EST片段 ,利用芯片拼接 (insilicocloning)的方法获得 2条新的带有完整开放阅读框 (ORF)的全长cDNA .利用生物信息学方法分析其同源性、二级结构、功能模体(motif)、保守区、结构域和家族分布情况 ,并且对这 2条新全长cDNA进行部分克隆测序鉴定 ,用半定量RT PCR实验证实其差异表达的真实性 .结果表明 ,这 2条全长cDNA为新基因 (GenBankAY0 35 2 12 ,AY0 35 2 13) ,它们在PTPα诱导表达前后确实存在差异表达 ,并与细胞能量代谢及酶功能相关 。An inducible protein tyrosine phosphatase α(PTPα)overexpression system, which could be used as a model for studying mechanism of tumorigenesis at early stage, was used to screen novel genes in NIH3T3 cells overexpressing PTPα. 24 hours after the overexpression of PTPα was induced, 65 differential bands were obtained, among which 29 known genes, 12 known ESTs, 6 novel ESTs were identified by bioinformatic analysis. Two novel full-length cDNAs with entire open reading frame(ORF) were obtained from ESTs by in silico cloning method. The two novel cDNAs were analyzed bioinformatically for homology, secondary structure, motif, domain and distribution of families and identified by semi-quantitative RT-PCR and sequencing. The results verified that the two full-length cDNAs were novel genes (GenBank AY035212, AY035213). The expression levels of the novel genes were changed after the oveexpression of PTPα was induced and their functions might be related to the enzymes involved in energy metabolism of cells. The results provide basis for further exploring the molecular mechanism of tumorigenesis and these genes may be candidate targets for gene therapy.

关 键 词：蛋白质酪氨酸磷酸酶α NIH3T3细胞恶变 肿瘤 早期形成机理 基因 鉴定 功能分析 DDRT-PCR 生物信息学 

分 类 号：R730[医药卫生—肿瘤] Q75[医药卫生—临床医学]