Hoxa10真核表达载体增强K562细胞对柔红霉素敏感度的研究  

Study on Hoxa10 Eukaryotic Expression Vector Increasing Sensitivity of K562 Cells to Daunorubicin

作　　者：范文文[1] 贾秀红[1] 李建厂[1] 

机构地区：[1]滨州医学院附属医院儿科,山东滨州256603

出　　处：《肿瘤防治研究》2012年第12期1424-1427,共4页Cancer Research on Prevention and Treatment

基　　金：山东省自然科学基金资助项目(Y2007C018);山东省科学技术发展计划资助项目(2010GSF10264)

摘　　要：目的构建高效干扰Hoxa10基因表达的shRNA真核载体,探讨其对人慢性髓系白血病细胞株K562对化疗药物柔红霉素(daunorubicin,DNR)敏感度的影响。方法设计合成针对Hoxa10的特异性shRNA寡核苷酸链,构建真核表达载体pGPHI/GFP/Neo-Hoxa10并测序,应用阳离子脂质体转染K562细胞。实验分三组:正常对照组、阴性对照组、实验组(分别为正常K562细胞、阴性对照质粒转染K562细胞、pGPHI/GFP/Neo-Hoxa10转染K562细胞),三组细胞均加入不同浓度的DNR。RT-PCR检测Hoxa10 mRNA的表达,应用MTT检测各组细胞对DNR的敏感度,流式细胞术检测细胞凋亡率。结果成功构建pGPHI/GFP/Neo-Hoxa10载体并转染K562细胞,该载体能有效降低Hoxa10mRNA表达水平ODR=(38.864±4.488)%;MTT结果显示Hoxa10重组载体可显著降低K562细胞对DNR的IC50(P<0.05),对DNR的敏感度能提高约3.58倍。流式细胞术结果显示,该载体下调Hoxa10的表达后,细胞的凋亡率显著增加,可达(16.207±4.891)%(P<0.05),且联合DNR后细胞凋亡率明显提高,凋亡率达(76.887±0.967)%(P<0.05)。结论本实验构建的靶向Hoxa10的真核表达载体对K562细胞中Hoxa10的表达有明显抑制作用,并能明显增强其对DNR的化疗敏感度。Objective To investigate the effect of eukaryotic expression vector mediated RNAi targeting Hoxal0 on the sensitivity of human Chronic Myeloid Leukemia K562 Cell line to daunorubicin. Methods shRNA oligo was designed and compounded according to the effective and specific siRNA strand from our previous test. Then plasmid pGPHI/GFP/Neo-Hoxal0 targeting Hoxal0 was constructed and sequenced. It was transfected into K562 cells by positive ion liposome. This experiment was divided into three groups ： normal control group, negtive control group and experimental group （normal K562 cells, negative control plasmid transfect K562 cells and pGPHI/GFP/Neo-Hoxa10 transfect K562 cells）. Different concentrations of DNR were added into these three groups, respectively. The drug sensitivity to DNR was de tected by MTT assay. The apoptosis changes were detected by flowcytometry. Results The plasmid pGPHI/GFP/Neo-HoxM0 was successfully constructed and transfected into K562 cells. The mRNA of Hoxal0 was inhibited by this plasmid in K562 cells by optical density rate = （38. 864 ±4. 488）%. The IC50 in the experimental group to DNR was significantly reduced as compared with that in the control（P〈 0. 05） ;and the sensitivity of K562 cells to DNR was elevated about 3.58 times. The apoptosis morphological rate of experimental group was（16. 207 ± 4. 891） %, which was significantly different from that of the control groups（P〈0.05） ;and the apoptosis morphological rate increased obviously after this vector combining DNR（P 〈0. 05）. Conclusion The expression of Hoxal0 can be effectively silenced by the eukaryotic expression vector pGPHI/GFP/Neo-Hoxal0,which can increase the sensitivity of K562 to DNR.

关 键 词：HOXA10 SHRNA 柔红霉素 K562 白血病 

分 类 号：R733.7[医药卫生—肿瘤]