摘要
目的探讨 mdr1短发夹 RNA(mdr1 shRNA)对人红白血病耐阿霉素细胞系 K562/ADM的耐药逆转作用。方法编码设计合成靶位 mdr1基因 mRNA 具有19个碱基发夹结构互补的寡核苷酸模板,构建2个 shRNA 表达载体 pSilencer^(TM)3.1-H1 neo mdr1-A和 mdr1-B,将其稳定转染 K562/ADM细胞。用 RT-PCR 法检测转染后 K562/ADM 细胞 mdr1 mRNA 表达,Western blot 检测 P-糖蛋白(P-gp)表达,流式细胞术和 MTT 法分别检测 K562/ADM 细胞凋亡和对阿霉素的敏感性,用激光共聚焦荧光显微镜观察并测定细胞内柔红霉素的积累。结果在 pSilencer^(TM)3.1-H11 neo mdr1-A 和 mdr1-B shRNA表达载体稳定转染的 K562/ADM 细胞,mdr1 mRNA 表达分别减少到转染前的35.9%(P<0.05)和27.5%(P<0.01);同时 Western blot 结果显示 P-gp 表达被明显而特异地抑制,对阿霉素的耐药性由79倍分别减低到38倍和30倍;并且,细胞内荧光强度与对照组相比显著增加(P<0.05),与阿霉素联合应用凋亡细胞百分率分别增加至18.1%(P<0.05)和54.4%(P<0.01)。结论靶向 mdr1基因shRNA 表达载体可有效逆转耐药,使耐药的肿瘤细胞恢复对化疗药物的敏感性。
Objective To explore the role of reversal multidrug resistance（MDR） using short hairpin RNA（shRNA） expression vectors in multidrug resistance human leukemia cell line K562/ADM. Methods The oligonucleotides with 19-mer hairpin structure were synthesized. The shRNA expression vectors were constructed and introduced into K562/ADM cells. Expression of mdr1 mRNA was assessed by RT-PCR, and P- gp expression was determined by Western blot. The apoptosis and sensitivity of the K562/ADM cells to doxorubicin were quantified by flow cytometry and methyl thiazolyl tetrazolium （MTT） assays, respectively. Cellular dannorubicin accumulation was assayed by laser confocol scanning microscope （ LCSM ）. Results In positive clones of K562/ADM cells stably transfected with pSilencer^TM 3.1-HI neo mdr1-A and mdr1-B shRNA expression vectors, RT-PCR showed that mdr1 mRNA expression was significantly reduced to 35.9 % （ P 〈0.05 ）, 27.5% （ P 〈 0.01 ）, respectively. Western blot showed that P-gp expression was significantly and specifically inhibited. Resistance against doxorubicin was decreased from 79-fold to 38-fold （ P 〈 0.05）, 30-fold （P 〈0.01 ） respectively. Furthermore, the fluorescence intensity of K562/ADM cells was increased significantly compared with the control, shRNA vectors significantly enhanced the cellular daunorubicin accumulation. The percent of the apoptosis cell was significantly enhanced to 18.1% （P 〈 0.05 ）, 54.4% （P 〈 0.01 ） respectively. Conclusions shRNA expression vectors can effectively reverse MDR, and restore the sensitivity of drug-resistance K562/ADM cells to conventional chemotherapeutic agents.
出处
《中华血液学杂志》
CAS
CSCD
北大核心
2007年第6期383-387,共5页
Chinese Journal of Hematology
