慢病毒介导shRNA稳定干扰P210^(bcr/abl)基因表达的体内外研究  

Stable Interference on P210^(bcr/abl) Gene Expression by Lentiviral Vectordelivered shRNA In Vitro and In Vivo

作　　者：朱玉峰[1] 王元占[1] 孟凡义[2] 

机构地区：[1]南方医科大学南方医院实验动物研究中心,广东广州510515 [2]南方医科大学南方医院实验动物研究中心血液病科,广东广州510515

出　　处：《中国实验血液学杂志》2012年第5期1090-1094,共5页Journal of Experimental Hematology

基　　金：广东省科技计划项目资助课题(编号2005B60302005)

摘　　要：P210bcr/abl融合基因在慢性髓系白血病(CML)发生、发展中起重要作用。针对P210bcr/abl融合基因的小分子抑制剂(如伊马替尼)治疗CML非常有效,但容易产生耐药性。P210bcr/abl基因的融合区为RNA干扰其表达提供了一个特异性靶点。本研究通过病毒载体建立能同时表达P210bcr/ablshRNA和P210bcr/abl基因的BaF3细胞系,并探讨慢病毒介导的shRNA对P210bcr/abl基因表达的作用。用荧光显微镜检测慢病毒对BaF3细胞的感染率,用台盼蓝拒染法检测细胞的增殖能力;用RT-PCR和Western blot分别检测P210bcr/ablmRNA和蛋白的表达。结果发现,P210bcr/ablshRNA能使BaF3-P210bcr/abl细胞中的P210bcr/ablmRNA及蛋白表达明显下降,并且能增强BaF3-P210bcr/abl细胞对伊马替尼的敏感性,与不表达P210bcr/ablshRNA的BaF3-P210bcr/abl细胞相比,伊马替尼对该细胞系的IC50下降50%以上。将该细胞由尾静脉移植到受致死剂量照射的裸小鼠体内,移植该细胞的小鼠生存期较移植BaF3-P210bcr/abl细胞的小鼠生存期明显延长。结论:稳定表达针对P210bcr/abl基因融合区的shRNA可能增强CML常规治疗的效果。P210bcr/abl fusion gene is indispensable for generation and progression of chronic myeloid leukemia（CML）.Small molecule inhibitors,such as imatinib,are effective for P210bcr/abl gene mediated CML,but drug resistance may occur.The unique fusion junction of P210bcr/abl gene is an attractive target for therapeutic intervention using RNA interference（RNAi）.This study was purposed to constructed the BaF3 cell line by viral vector which can stably express P210bcr/abl shRNA and P210bcr/abl mRNA at the same time,and investigate the effect of lentiviral-victor-delivered shRNA on P210bcr/abl gene expression.The infective rate of lentiviral vector on BaF3 cells with P210bcr/abl gene was assayed by fluorescent microscopy;the cell proliferation ability was determined by trypan blue exclusion;the P210bcr/abl mRNA and protein expressions were detected by RT-PCR and Western blot respectively.The results found that stable expression of the P210bcr/abl shRNA resulted in obvious inhibition of P210bcr/abl mRNA and protein expression and increased sensitivity of these P210bcr/abl gene transformed Ba/F3 cells to imatinib.The IC50 to imatinib in these cells decreased50% as compared with Ba/F3-P210bcr/abl cells which did not express P210bcr/abl mRNA.The survival time of the lethal dose irradiated mice induced by intravenous injection of these Ba/F3 cells was longer than the other group induced by Ba/F3-P210bcr/abl.It is concluded that stable expression of shRNA targeting the P210bcr/abl gene fusion junction may potentiate the effects of conventional therapy for CML.

关 键 词：慢性髓系白血病 P210bcr/abl融合基因 RNA干扰 慢病毒载体 

分 类 号：R733.72[医药卫生—肿瘤] Q7[医药卫生—临床医学]