银染RNA AP-PCR差示法克隆人胃癌转移相关基因  

Molecular Cloning of Human Stomach Carcinoma Metastasis Related Genes by Silver Stained RNA AP PCR Differential Display

作　　者：陈瑞川[1] 蔡克瑕[1] 苏金华[1] 马胜平[2] 付永贵[2] 吴艳环[3] 

机构地区：[1]厦门大学生命科学学院抗癌研究中心,福建厦门361005 [2]厦门大学生命科学学院生物学系,福建厦门361005 [3]厦门市中山医院消化科,福建厦门361003

出　　处：《癌症》2001年第11期1267-1271,共5页Chinese Journal of Cancer

基　　金：福建省自然科学研究基金项目(No:F00024)

摘　　要：目的:以改进的银染RNAAP-PCR法分析原发性及转移性胃癌标本,鉴定和克隆胃癌转移相关基因。方法:以原发及转移灶胃癌标本总RNA为研究对象,首先以T12MN“锚定”引物进行逆转录合成cDNA第一链,再以10核苷酸任意引物进行PCR扩增,5%非变性聚丙烯酰胺凝胶电泳分离扩增产物后银染显示并回收差异条带,经RNA点杂交验证、克隆测序后进入GenBank数据库检验同源性。结果:经银染RNAAP-PCR法分析,获得系列差异表达片段。对其中MGA1(662bp)和PGA1(589bp)两个片段进行验证和测序,结果显示,MGA1与胃癌转移相关,并与编码人巨噬细胞集落刺激因子受体(macrophagecolony-stimulatingfactorreceptor,CSF-1R)的原癌基因c-fms100%同源;而PGA1则属胃癌转移抑制基因,与编码肿瘤转移抑制基因KAI-1完全同源。结论:银染RNAAP-PCR法适用于分析和克隆差异表达基因,具有快速、直观、假阳性率低等优点;提示CSF-1R及KAI-1与胃癌转移表型相关,为进一步研究CSF-1R功能提供了新的线索。Objective: To identify and clone differentially expressed genes in human primary and metastatic stomach carcinomas with improved silver stained RNA arbitrarily primed PCR method (RNA AP PCR). Methods: The total RNA populations which were respectively extracted from primary or metastatic stomach carcinoma tissue were firstly reverse transcribed with 'anchor primer' olig(dT)12MN. Synthesized cDNA pools were then subjected to AP PCR amplification with 10 nt arbitrarily primers. Amplified products were resolved by electrophoresis on non denatural polyacrylamide gel and the differentially expressed PCR fragments were separated from silver stained gel. After RNA hybridization, T A vector cloning and DNA sequencing, the DNA sequences of fragments were checked with GenBank data for homology. Results: With improved silver stained RNA AP PCR, several differentially expressed PCR fragments were separated from primary or metastatic stomach carcinoma sample. Two fragments MGA1(662 bp) and PGA1 (589 bp) were reamplified and finally sequenced. The results showed that MGA1 was highly expressed in metastatic stomach carcinoma tissues and is complete homology to oncogene c fms, a gene coding human macrophage colony stimulating factor receptor (CSF 1R). PGA1, highly expressed in primary stomach carcinoma tissues, is 100%homology to tumor metastasis suppressor gene KAI 1. Conclusions: The improved silver stained RNA AP PCR method is suitable for identification and cloning differentially expressed genes. The results suggest that CSF 1R may be related to the metastatic phenotype of stomach carcinoma.

关 键 词：AP-PCR银染 胃肿瘤 转移相关基因 

分 类 号：R735.2[医药卫生—肿瘤]