摘要
目的探讨沉默Delta-like ligand 3(DLL3)基因对白血病K562/ADM细胞对阿霉素(ADM)耐药性的影响及其分子机制。方法将干扰人DLL3基因表达的shRNA质粒和无义对照质粒转染K562/ADM细胞,采用RT-PCR法检测DLL3的mRNA表达水平,采用CCK-8法检测ADM对K562和K562/ADM细胞的毒性作用,采用流式细胞术检测细胞凋亡和细胞内ADM浓度,采用Western blotting方法检测DLL3、谷胱甘肽S转移酶-π(GST-π)和P-糖蛋白(P-gp)的蛋白表达水平。结果 K562/ADM细胞DLL3的mRNA和蛋白表达水平均显著高于其亲代K562细胞(P <0. 05); ADM对K562和K562/ADM细胞的IC50分别为1. 08 mg/L和34. 93 mg/L;沉默DLL3基因后,K562/ADM细胞的耐药倍数下降至13. 12,反转倍数为2. 47;尽管抑制DLL3基因表达未对K562/ADM细胞凋亡产生影响,但可下调P-gp和GST-π的蛋白表达水平(P <0. 05),增加K562/ADM细胞内ADM的蓄积量(P <0. 05),从而增强ADM诱导的K562/ADM细胞凋亡(P <0. 05)。结论沉默DLL3基因可反转K562/ADM细胞对ADM的耐药性,这可能与下调P-gp和GST-π蛋白水平、从而减少K562/ADM细胞内阿霉素蓄积量有关。
Objective To investigate whether Delta-like ligand 3（ DLL） gene silencing could influence the sensibility of human leukemia K562/ADM cells to adriamycin（ ADM） in vitro. Methods K562/ADM cells were stably transfected with specific shRNA interference plasmid vector targeting for DLL3. The mRNA expression level of DLL3 were measured by RT-PCR. CCK-8 assay was employed to detect the cytotoxic effect of ADM in K562 and K562/ADM cells.The apoptosis and intracellular ADM concentration were analyzed by flow cytometry. Western blotting was performed to determine the protein expression levels of DLL3,glutathione S transferases-π,（ GST-π） and P-glycoprotein（ P-gp）.Results K562/ADM cells had a significantly higher mRNA and protein expression level of DLL3 than K562 cells（ P〈0. 05）. The IC50 value of K562 and K562/ADM cells to ADM were 1. 08 and 34. 93 mg/L respectively. After DLL3 gene silencing,the resistant factor of K562/ADM cells declined to 13. 12 with a reversal fold of 2. 47. DLL3 gene silencing had no effect on apoptosis of K562/ADM cells,but a significant effect on down-regulating protein levels of P-gp and GST-π（ P〈0. 05） and increasing intracellular ADM concentration（ P〈0. 05）,and resulting in an enhance of ADM-induced apoptosis（ P〈0. 05）. Conclusion DLL3 gene silencing may enhance the sensibility of K562/ADM cells to ADM by down-regulating P-gp and GST-π,and increasing intracellular ADM concentration.
作者
王世广
司旭艳
王丽君
仝雷
李阳杰
WANG Shi-guang;SI Xu-yan;WANG Li-jun;TONG Lei;LI Yang-jie(Department of Medical Function,Medical College,Zhengzhou University of Industrial Technology,He＇nan Xinzheng 451150,China;Department of Clinical Foundation,Medical College,Zhengzhou University of Industrial Technology,He＇nan Xinzheng 451150,China;Department of Medical Morphology,Medical College,Zhengzhou University of Industrial Technology,He＇nan Xinzheng 451150,China;Department of Pharmacy,Medical College,Zhengzhou University of lndustrial Technology,He＇nan Xinzheng 451150,China)
出处
《解剖学报》
CAS
CSCD
北大核心
2018年第5期624-629,共6页
Acta Anatomica Sinica
