CAJ | 학술논문

目的研究糖原合成酶激酶-3β(glycogen synthase kinase-3β,GSK-3β)在骨肉瘤细胞增殖中的作用及相关分子机制,探讨其在骨肉瘤靶向治疗中的价值.方法 Western blot检测人成骨细胞及骨肉瘤细胞p-GSK-3β (Ser9)表达水平,观察GSK-3β抑制剂及siRNA干扰对细胞增殖、凋亡的影响,利用凋亡蛋白芯片筛查并验证GSK-3β调控骨肉瘤细胞的分子机制,通过裸鼠体内实验评估靶向GSK-3β对于骨肉瘤的治疗价值.结果在骨肉瘤细胞中p-GSK-3β (Ser9)表达水平明显低于成骨细胞.GSK-3β特异性抑制剂及siRNA 干扰均可明显抑制骨肉瘤细胞增殖并诱导凋亡蛋白cleave-caspase 3表达量明显上调.通过蛋白芯片筛查显示一组NF-κB调控的靶基因蛋白survivin、cIAP-1、XIAP及Bcl-2明显下调.Western blot验证了上述芯片结果,并发现氯化锂处理后p-IκBo水平下降,核内NF-κB p65表达量下降.NF-κB双荧光素酶报告系统检测稳定过表达持续活化GSK-3β细胞中NF-κB转录活性与空载体组细胞相比明显升高,而稳定干扰GSK-3β的细胞中NF-κB转录活性与空载体组细胞相比明显下降.裸鼠体内动物实验发现稳定干扰GSK-3β和采用氯化锂均可明显抑制骨肉瘤皮下移植瘤的生长.结论 GSK-3β在骨肉瘤中处于相对活化状态,可通过上调NF-κB转录活性调控骨肉瘤细胞增殖；靶向GSK-3β是骨肉瘤潜在的治疗新策略.
목적 연구당원합성매격매-3β(glycogen synthase kinase-3β,GSK-3β)재골육류세포증식중적작용급상관분자궤제,탐토기재골육류파향치료중적개치.방법 Western blot검측인성골세포급골육류세포p-GSK-3β (Ser9)표체수평,관찰GSK-3β억제제급siRNA간우대세포증식、조망적영향,이용조망단백심편사사병험증GSK-3β조공골육류세포적분자궤제,통과라서체내실험평고파향GSK-3β대우골육류적치료개치.결과 재골육류세포중p-GSK-3β (Ser9)표체수평명현저우성골세포.GSK-3β특이성억제제급siRNA 간우균가명현억제골육류세포증식병유도조망단백cleave-caspase 3표체량명현상조.통과단백심편사사현시일조NF-κB조공적파기인단백survivin、cIAP-1、XIAP급Bcl-2명현하조.Western blot험증료상술심편결과,병발현록화리처리후p-IκBo수평하강,핵내NF-κB p65표체량하강.NF-κB쌍형광소매보고계통검측은정과표체지속활화GSK-3β세포중NF-κB전록활성여공재체조세포상비명현승고,이은정간우GSK-3β적세포중NF-κB전록활성여공재체조세포상비명현하강.라서체내동물실험발현은정간우GSK-3β화채용록화리균가명현억제골육류피하이식류적생장.결론 GSK-3β재골육류중처우상대활화상태,가통과상조NF-κB전록활성조공골육류세포증식；파향GSK-3β시골육류잠재적치료신책략.
Objective To study the affect and the related molecular mechanism of glycogen synthase kinase-3β in the proliferation of osteosarcomaand its value in the target therapy of osteosarcoma.Methods The expression level of p-GSK-3β(Ser9)and GSK-3β were detected in human osteoblast cell and osteosarcoma cells by western blot.Observe the effect of GSK-3β inhibitors and siRNA interference on the GSK-3β regulate osteosarcoma cells using apoptosis protein chip.Evaluate the valueof GSK-3β target therapy on osteosarcoma in vivo.Results The expression level of p-GSK-3β (Ser9)was lower in osteosarcoma cells.LiCL,GSK inhibitor Ⅸ,siRNA knockdown could inhibit the cell viability and up-regulated the apoptosis-related protein cleaved-caspase3.The results of the protein array showed that downstream proteins of NF-κB downregulated significantly.The results were validated by western blot,while the downregulation of p-Iκ-Bα and nuclear NF-κB p65 were also observed after LiCL treatment.Inhibition of GSK-3β by either LiCl or specific siRNA resulted in a significant reduction of NF-κB luciferase reporter activity.Furthermore,the NF-κB luciferase reporter activity was significantly increased in CA cell lines,but not in KD cell lines.By contrast,NF-κB-luciferase reporter activity was significantly decreased in stably GSK-3β knockdown cells.GSK3β inhibitor LiCL and shRNA knock down demonstrated a strong cytotoxicity effect on osteosarcoma cells in vivo.Conclusion GSK-3β is in the state of relative active in osteosarcoma in osteosarcoma and important in cell proliferation.GSK-3β regulates cell survival partially through the NF-κB pathway.It is a promising therapeutic target in osteosarcoma.