抑制性差减杂交技术分离白血病多药耐药基因  

Multidrug resistance associated genes of leukemia separated by suppression subtractive hybridization

作　　者：王楠[1] 潘喆[2] 袁宏[1] 

机构地区：[1]大连医科大学附属第一医院检验科,辽宁大连116023 [2]大连医科大学附属第一医院病理科,辽宁大连116023

出　　处：《国际检验医学杂志》2016年第6期743-745,748,共4页International Journal of Laboratory Medicine

摘　　要：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RsaⅠ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。Objective To isolate and identify differential expression genes associated with multidrug resistance of leukemia.Methods Differential expression genes between leukemia cell line K562 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 restriction enzyme RsaⅠ,then connected with adopter1 and adopter2R,and linked with pMD19-T vector.Constructed vectors were transferred 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.

关 键 词：白血病 多药耐药 抑制性差减杂交 差异表达基因 

分 类 号：R730.43[医药卫生—肿瘤]