摘要
目的应用小分子干扰RNA(siRNA)技术抑制survivin基因的表达,观察其对K562细胞化疗敏感性的影响,为临床白血病治疗提供理论依据。方法实验分为RNA干扰组、脂质体对照组、空白对照组。转染48h后用RT-PCR法,免疫组化法分别检测survivin在mRNA及蛋白水平的表达;MTT法检测细胞对阿霉素的敏感性;流式细胞仪检测细胞内阿霉素的药物浓度;免疫组化法检测K562细胞中3种耐药相关蛋白P-gp、TOPO-2、GST-π的表达。结果①RNA干扰组K562细胞的survivin基因表达水平明显下降;②RNA干扰组K562细胞对阿霉素敏感性增高;③RNA干扰组K562细胞中阿霉素浓度增高;④RNA干扰组K562细胞耐药蛋白GST-π表达水平下降,而P-gp,TOPO-2的表达与对照组比较无显著差异。结论特异性siRNA能够有效抑制survivin基因的表达,并可能通过降低耐药蛋白GST-π的表达增加K562细胞对阿霉素的敏感性。
Objective To explore the effects of survivin-targeted small interference RNA （siRNA） on the sensitivity of K562 cell line to adriamycin （ADM）. Methods K562 cells were divided into three groups： RNA interference （RNAi） group, lipofectamine group and control group. Cell transfection was induced by lipo- fectamine2000. The expressions of survivin in I〈562 cells were assayed 48 h after transfection by RT-PCR and immunohistochemical methods. The protein levels of P-glycoprotein （P-gp）, TOPO-2, GST-π were detected using immunohistochemical methods. The sensitivity of K562 cells to ADM was determined by MTT and flow cytometry. Results After transfection with recombinant plasmid pshRNA-survivin, the mRNA and protein expressions of survivin in K562 cells were decreased significantly （ P 〈 0. 05 ）. The sensitivity of K562 cells to ADM was increased in RNAi group as compared with lipofectamine group and control group （P 〈 0. 05）. The protein level of GST-π expression was significantly lower in RNAi group than those in the other two control groups （P 〈 0. 05）. Conclusion Survivin-targeted RNA interference can increase the sensitivity of K562 cells to ADM, probably by decreasing the expression of GST-π.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2008年第22期2111-2114,共4页
Journal of Third Military Medical University
基金
重庆市教委科研基金(X3004-020050033)~~
