应用抑制性消减杂交技术克隆和筛选白血病多药耐药相关基因

Identification of multidrug resistance related genes in leukemia by suppression subtractive hybridization

目的 克隆和筛选白血病多药耐药 (MDR)相关基因。方法 以MDR白血病细胞株K5 6 2 DOX为检测样本 ,以K5 6 2细胞株为驱赶样本 (对照 ) ,应用抑制性消减杂交 (SSH)技术分离和克隆K5 6 2 DOX细胞中过高表达的基因 ,构建cDNA消减文库 ;通过反向Northern斑点杂交法进一步筛选消减文库 ,对阳性克隆进行序列测定和同源性分析 ;并采用RT PCR和Northernblot方法对某些阳性克隆进行进一步的验证。结果 发现了 11个在K5 6 2 DOX细胞中高表达的基因 ,包括S3核糖体蛋白 (S3ribosomalprotein ,S3rp)基因、尼克酰胺腺嘌呤二核苷酸脱氢酶亚单位 2 (NADHdehydrogenasesubunit2 ,ND2 )基因、My0 2 3基因等 ,其中多数基因与肿瘤MDR的关系尚未见报道。结论 筛选到几个可能与白血病MDR形成有关的基因 ,提示了白血病MDR发生的新机制。

Objective To clone and screen genes related to multidrug resistance(MDR) in leukemia. Methods Suppression subtractive hybridization(SSH) was performed to profile differentially expressed genes between a MDR leukemia cell line (K562/DOX, as tester) and its parent cell line (K562, as driver). Reverse Northern dot blot was carried out to further screen the subtracted cDNA library. The overexpressed cDNA fragments in K562/DOX cells were sequenced and compared with known genes in Genbank. RT-PCR and Northern blot were employed to confirm the differential expression of some identified genes. Results Eleven genes were identified being overexpressed in K562/DOX, including S3 ribosomal protein (S3rp)gene, NADH dehydrogenase subunit 2(ND2) gene and My023 gene, which have not been reported to be related to MDR in cancer. Conclusion Several genes, which might be involved in MDR were identified, indicating novel mechanisms of MDR in leukemia.