c-cbl反义RNA抑制K562细胞的增殖  

Antisense RNA of proto-oncogene c-cbl inhibits proliferation of K562 leukemic cells

作　　者：郭子宽[1] 李金兰[1] 高晖[1] 付家瑜[1] 陈珊珊[1] 

机构地区：[1]北京医科大学人民医院血液病研究所,北京100034

出　　处：《中国实验血液学杂志》1998年第2期140-144,共5页Journal of Experimental Hematology

摘　　要：为探讨原癌基因c-cbl对CML细胞增殖的影响,我们用RT-PCR克隆了包括5′端非编码区的人类c-cbl基因部分序列,将之反向插入pcDNA3载体,并转染K562及NB4细胞。经MTT实验、半固体集落培养和流式细胞术观察细胞增殖能力的改变。结果表明,限制性酶切分析及序列测定证明所克隆的基因核苷酸序列正确,MTT实验显示反义载体转染K562细胞生长明显受抑,24、48及72小时增殖抑制率分别为30.07％,42.53％和55.75％(P值均<0.001),FCM-BrdU法测定的细胞倍增时间之比为1∶1.87(P<0.02),细胞集落形成抑制率为33.01％,反义转染基因NB4细胞增殖率无改变。上述结果直接地证明了c-cbl在CML细胞生长中的重要性。To clarify the effect of proto-oncogene c-cbl in CML celis, 5' portion of c-cbl gene was cloned by RT-PCR and then transfected into K562 and NB4 cells in the antisense orientation with lipofectin. MTT test, colony forming efficiency and flow cytometric BrdU labelling assay were performed to evaluate their proliferative status. Restriction enzyme digestion and DNA sequencing showed that the cloned fragment corresponded completely to published sequence of human c-cbl. The transfected K562 celis proliferated at a much slower rate than control cells as evaluated by MTT test and FCM-BrdU technique. Growth inhibition res were 30.06%, 42. 53% and 55.75% at 24, 48 and 72 hr respectively. The potential doubling time (Tpot) of K562 celis transfected antisense was almost two times longer than that of control ( P < 0.02). The ratio of colony forming efficiency was 1:1.48 (P<0.001). However, antisense transcripts to c-cbl had no proliferation inhibition effect on NB4 cells. The results here demonstrated that onco-protein P120CBL is critical for the growth of Ph+ cells, and it may play an importmant role in the signaling network activated by P210BCR/ABL.

关 键 词：反义RNA 原癌基因 c-cbl基因 K562细胞 Ph^+人白血病细胞系 慢性粒细胞白血病 

分 类 号：Q753[生物学—分子生物学] R733.72[医药卫生—肿瘤]