抑制性差减杂交技术分离白血病多药耐药基因

摘要

Objective To isolate and identify differential expression genes associated with multidrug resistance of leukemia . Methods Differential expression genes between leukemia cell line K 562 and resistant cell lines K562/DOX were isolated by using suppression subtractive hybridization (SSH) technique .Total RNA were extracted .cDNA were synthesized and digested by restric-tion enzyme Rsa Ⅰ ,then connected with adopter1 and adopter2R ,and linked with pMD19-T vector .Constructed vectors were trans-ferred into E .coli .Subtracted cDNA library was constructed ,and the positive clones were screened according to base sequences and homologous sequences .The differential expression genes were indentified by comparison analysis of Gene Bank database .Results A total of 220 differential expression genes were sequenced ,including hemoglobin ,ribosomes and mitochondria related genes ,and heat shock factor binding protein 1 (HSPB1) gene and other genes .Conclusion SSH method and molecular cloning technique could be used to construct subtracted cDNA library of differential expression genes between drug resistant and not -resistant leukemia cells , which might be useful for further screening and cloning of differential expression genes of multidrug resistant tumor cells .%目的：分离及鉴定与白血病多药耐药性相关的差异表达基因。方法采用抑制性差减杂交（SSH）技术分离非耐药细胞株 K562与耐药细胞株 K562／DOX 差异表达基因。提取总 RNA ，逆转录合成 cDNA ，经限制性内切酶 Rsa Ⅰ酶切后，分别与不同的接头（adopter1和 adopter2R）连接；连接产物插入 pMD19-T 载体后转入大肠埃希菌中，构建 cDNA 差减文库；挑取阳性克隆提取质粒进行测序及同源序列分析，确定差异表达基因。结果筛选获得220个差异表达基因，包括血红蛋白、核糖体和线粒体等相关基因，以及热休克因子结合蛋白（HSPB1）基因等其他基因。结论采用 SSH 技术及分子克隆技术可构建耐药及非耐药肿瘤细胞株差异表达基因的差减 cDNA 文库，能够为进一步筛选、克隆肿瘤细胞多药耐药性相关差异表达基因奠定基础。