p16ink4a/hRb1对骨肉瘤细胞周期协同的调控作用  

Effect of exogenous p16ink4a and hRbl genes on cell cycle regulation of osteosarcoma cell line

作　　者：廖翔[1] 杨述华[1] 邵增务[1] 李进[1] 刘勇[1] 熊小芊[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院骨科,武汉430022

出　　处：《中华实验外科杂志》2006年第6期728-730,共3页Chinese Journal of Experimental Surgery

基　　金：湖北省自然科学基金(2005ABA151)

摘　　要：目的观察外源性p16ink4a/hRb1基因联合导入骨肉瘤细胞后,对其细胞周期的协同调控作用。方法利用本室构建的pIRES-p16ink4a-hRb1、pIRES-p16ink4a与pIRES-hRb1质粒,脂质体介导转染p16缺失,hRb1表达阳性的骨肉瘤细胞株MC-63,G418筛选获得抗性克隆,通过逆转录-聚合酶链反应(RT-PCR)和Western blot半定量分析外源基因表达；Sub G_1法流式细胞术分析细胞周期特异性和凋亡率。噻唑蓝(MTT)比色法与细胞生长曲线观察细胞增殖情况。结果外源基因在靶细胞mRNA和蛋白水平分别有独立表达。与对照组比,所有外源性基因导入组细胞周期均显著阻滞在G_1期(P＜0．01)并存在凋亡,且双基因导入组凋亡率分别比单基因组高4．04和6．94倍(P＜0．01)；所有外源性基因导入组各时间点细胞生长抑制率均显著上升,且双基因导入组细胞高于单基因组(P＜0．01)。结论p16ink4a/hRb1联合导入骨肉瘤细胞后,干扰了p16ink4a-Cy- clinD1/CDK-hRb1负反馈循环,比单基因导入更能抑制肿瘤细胞生长和杀灭肿瘤细胞。Objective To study the effect on regulation of cell cycle of osteusarcoma cell line MG63 by exogenous p16ink4a and hRb1 genes. Methods pIRES-p16ink4a-hRb1, pIRES-p16ink4a and pIRES-hRb1 plasmids were reserved by our laboratory. The recombinant plasmid was transferred into osteusarcoma cell line MG63 by metafectene, and the resistant clones were selected by G418 selective medium. The mRNA and protein expression in osteusarcomacell line was assayed by RT-PCR and Westernblot respectively. Cell cycle and apoptosis were analyzed by subG1 flow cytometry. Cell proliferation was tested by MTT. Results The recombinant plasmid pIRES-p16ink4a-hRb1 was successfully constructed. In the genome of these transfected target cells, the expression of p16ink4a and hRb1 mRNA and protein was detected respectively in vitro, subG1 flow cytometric analysis and MTT demonstrated that combined used of p16ink4a and hRb1 genes more significantly inhibited cell growth and induced a more marked G1 arrest and apoptosis than p16ink4a/hRb1 alone.Conclusion Co-expression of exogenous p16ink4a with hRb1 broke the regulatory feedback loop of p16ink4a-cyclinD1/CDK-hRb1 and played more significant role in inhibiting cell growth as well as inducing cell apoptosis than p16ink4a or hRb1 did alone in vitro.

关 键 词：骨肉瘤 细胞周期 流式细胞术 

分 类 号：R738[医药卫生—肿瘤]