Liao Xiang, Yang Shuhua, Shao Zengwu, Li Jin, Liu Yong, Xiong Xiaoqian, Liu Xin
Department of Orthopedic, Union Hospital, Tongji Medical Collage, Huazhong University of Science and Technology, Wuhan 430022, China.
J Huazhong Univ Sci Technolog Med Sci. 2005;25(6):679-82. doi: 10.1007/BF02896169.
To study the effect on regulation of cell cycle of osteosarcoma cell line MG63 tranceduced with exogenous p16ink4a and hRb1 genes, pIRES-p16ink4a-hRb1, pIRES-p16ink4a and pIRES-hRb1 plasmids were constructed by gene recombination technology. The recombinant plasmid was transferred into osteosarcoma cell line MG63 by metafectene, and the resistant clones were selected by G418 selective medium. mRNA and protein expression of osteosarcoma cell line were assayed by RT-PCR and Western-Blot respectively. Cell cycle and apoptosis were analyzed by subG1 flow cytometric. Cell proliferation was tested by MTT. In the genome of these transfected target cells, the expression of p16ink4a and hRb1 mRNA and protein were detected respectively in vitro. It was demonstrated with subG1 flow cytometric analysis and MTT method that p16ink4a and hRb1 genes cooperation more significantly inhibited cell growth and induced a more marked G1 arrest and apoptosis than p16ink4a/hRb1 alone (P < 0.01). Coexpression of exogenous p16ink4a with hRb1 broke the regulatory feedback loop of p16ink4a-cyclinD1 /CDK-hRb1 and played a more significant role in inhibiting cell growth as well as inducing cell apoptosis than p16ink4a or hRb1 did alone in vitro.
为研究外源性p16ink4a和hRb1基因转导对骨肉瘤细胞系MG63细胞周期调控的影响，采用基因重组技术构建了pIRES-p16ink4a-hRb1、pIRES-p16ink4a和pIRES-hRb1质粒。通过metafectene将重组质粒转入骨肉瘤细胞系MG63，并用G418选择培养基筛选抗性克隆。分别采用RT-PCR和Western-Blot检测骨肉瘤细胞系的mRNA和蛋白表达。通过亚G1流式细胞术分析细胞周期和凋亡情况。采用MTT法检测细胞增殖情况。在这些转染的靶细胞基因组中，分别在体外检测p16ink4a和hRb1 mRNA及蛋白的表达。亚G1流式细胞术分析和MTT法结果表明，与单独的p16ink4a/hRb1相比，p16ink4a和hRb1基因联合作用更显著地抑制细胞生长，诱导更明显的G1期阻滞和凋亡（P < 0.01）。外源性p16ink4a与hRb1共表达打破了p16ink4a-cyclinD1 /CDK-hRb1的调节反馈环，在体外抑制细胞生长以及诱导细胞凋亡方面比单独的p16ink4a或hRb1发挥更显著的作用。
