改良消减杂交法克隆人乳腺癌MCF-7细胞凋亡相关基因

Apoptosis-related genes cloned by improved subtractive hybridization

目的 克隆人乳腺癌MCF 7肿瘤细胞凋亡相关的基因 ,分析所克隆基因与凋亡的关系。方法 用全反式维甲酸诱导人乳腺癌MCF 7细胞凋亡 ,建立肿瘤细胞凋亡模型 ;运用基于聚合酶链式反应 (PCR)的改良消减杂交技术 ,克隆肿瘤细胞凋亡的相关基因。结果 在筛选出的 5个特异表达的基因中 ,4个是已知基因 ,1个新基因。在 4个已知基因中 ,3个与凋亡关系密切 ,其中hsp 90和rb L3与凋亡的关系文献报道较少。结论 维甲酸所诱导的肿瘤细胞凋亡是一个多基因参与的过程 ,这种基于PCR的改良消减杂交技术为克隆新基因提供了一个新的方法和思路。

Objective To clone apoptosis related genes induced by all trans retinoic acid (ATRA) from human breast cancer cell line MCF 7, and to analyze the association between the cloned genes and apoptosis. Methods An apoptotic cell model of MCF 7 cells was constructed with ATRA induction. The apoptosis related genes were cloned by improved PCR based subtractive hybridization.Results Twelve differentially expressed clones were screened out. After exclusion of false positive clones by reverse dot blotting, 5 clones containing fragments of 0.5～1.5 kb were sequenced. The results of sequencing were compared with BLAST. A novel gene,named apmcf 1, coding for 47 amino acids was identified. This gene was accepted by GenBank, (Accession number: AF141882). The result of reverse dot blotting showed that it was related to apoptosis. The other 4 genes were already known. Three of them were related to apoptosis as previously reproted. Two of them, hsp 90 and rb L3, had little information about their relationship with apoptosis.Conclusion ATRA induced tumor cell apoptosis is a complex process with multiple genes involved.