摘要
目的 研究转染反义寡脱氧核苷酸 (antisenseoligode oxynucleotides, ASODN)对Cdk7特异性的沉默作用及其对体外培养人肝细胞癌HepG2细胞pRb和Cdk2磷酸化水平以及细胞周期进展的影响,确定Cdk7作为抗肿瘤药物研发靶点的可行性。方法 以免疫印迹检测转染ASODN对Cdk7的特异性沉默作用及其对pRb和Cdk2磷酸化水平的影响;以流式细胞术测定转染ASODN后细胞DNA含量,确定细胞周期进展和凋亡情况,透射电子显微镜观察细胞凋亡形态。结果 转染ASODN 72h后,Cdk7蛋白水平显著下降,有明显剂量 -效应关系,最高可降至对照组的 0 12±0 05;在ASODN浓度>100nmol L-1时,pRb和Cdk2磷酸化水平显著下降,两种蛋白的磷酸化水平与给药剂量有明显依赖关系。随转染ASODN浓度上升及转染时间的延长,G0 /G1 期细胞、凋亡细胞比例迅速升高,与对照组比较,出现明显的G0 /G1 期阻滞(P<0 01)和细胞程序性死亡(P<0 01);透射电子显微镜观察显示>50nmol·L-1各组细胞具特征性凋亡形态。结论 用ASODN沉默Cdk7可降低pRb以及Cdk2的磷酸化水平,使其生物活性下降,对体外培养HepG2细胞可产生显著的G0 /G1 期阻滞,引起细胞周期延长和细胞凋亡,产生抗增殖作用,可以Cdk7作为抗肿瘤药物研发的新靶点。
Aim To evaluate the effect of Cdk7 silencing on the c ell cycle control, the phosphorylation level changes of Cdk2 and pRb in human he patoblastoma HepG2 cell culture in vitro, and to validate Cdk7 as a novel ta rget for anticancer therapeutics.Method Levels of Cdk7 and the phosphorylation levels of Cdk2 and pRb were measured by Western-blotting. DNA c ontents, cell cycle and apoptosis induced by Cdk7 silencing were analyzed by flo w cytometry and ultrastructural changes of cells were observed with transmission electron microscopy.Result The phosphorylation levels of pRb a nd Cdk2 and the levels of Cdk7 decreased in a concentration-dependent manner wh en the concentration was above 100 nmol·L -1. Indice of cells arrested in G 0/G 1 phases and apoptotic cells increased in a dosage-and time-dependent manner, the difference was significant between Cdk7 ASODN and the sense control (P<0.01); Characteristic apoptosis in Cdk7 ASODN treated groups were obviou s under the transmission electron microscopy.Conclusion Bioacti vities and phosphorylation levels of pRb and Cdk2 decreased after Cdk7 silencin g and thus induced obvious G 0/G 1 phases arrest and apoptosis in HepG2 cell c ulture in vitro, it is feasible to consider Cdk7 as a novel target for antic ancer therapeutics.
出处
《中国药理学通报》
CAS
CSCD
北大核心
2005年第1期106-110,共5页
Chinese Pharmacological Bulletin
