论文部分内容阅读
目的:观察新型哺乳动物雷帕霉素靶蛋白(mammalia target of rapamycin,mTOR)抑制剂含磷西罗莫司衍生物FIM-A对人骨肉瘤MG-63细胞增殖及凋亡的影响。方法:不同浓度(1×10-9～1×10-5mol/L)FIM-A处理MG-63细胞后,采用CCK-8法检测MG-63细胞的增殖,流式细胞术检测MG-63细胞周期和凋亡情况,ELISA法检测血管内皮细胞生长因子(vascu-lar endothelial cell growth factor,VEGF)和低氧诱导因子(hypoxia inducible factor-1α,HIF-1α)的分泌量,RT-PCR和Western blot-ting分别检测FIM-A对MG-63细胞中mTOR、p70核糖体S6激酶(p70S6 kinase protein,p70s6k)及4E结合蛋白1(4E-bindingprotein 1,4E-BP1)mRNA和蛋白表达的影响。结果:与人成骨hF-OB1.19细胞相比,人骨肉瘤MG-63细胞中mTOR、p70s6k及4E-BP1 mRNA的表达水平明显升高(P<0.05)。FIM-A可有效抑制MG-63细胞的增殖(P<0.05),且呈剂量依赖性(r=0.940,P<0.01)。1×10-6mol/L FIM-A处理24 h后与对照组相比,G0/G1期MG-63细胞比例明显增加[(56.4±3.2)%vs(43.4±6.9)%,P<0.05],而MG-63细胞的凋亡率没有明显改变。不同浓度FIM-A作用24 h后,MG-63细胞中HIF-1α和VEGF表达均明显低于对照组(P<0.05),且具有剂量依赖性(HIF-1α,r=-0.988,P<0.01;VEGF,r=-0.998,P<0.01)。同时,FIM-A对MG-63细胞中mTOR(r=-0.919,P<0.01)、p70s6k(r=-0.843,P<0.01)及4EBP1(r=-0.818,P<0.01)蛋白的磷酸化也具有浓度依赖性抑制作用。结论:FIM-A能抑制人骨肉瘤MG-63细胞的增殖,并阻滞细胞周期于G0/G1期,其机制可能与影响mTOR信号通路蛋白磷酸化有关。
OBJECTIVE: To observe the effect of a novel mammalian target of rapamycin (mTOR) inhibitor of phosphorus-containing sirolimus derivative FIM-A on the proliferation and apoptosis of human osteosarcoma MG-63 cells. Methods: MG-63 cells were treated with different concentrations of FIM-A (1 × 10-9 ~ 1 × 10-5mol / L). The proliferation of MG-63 cells was detected by CCK-8 assay. The cell cycle and apoptosis were detected by enzyme-linked immunosorbent assay (ELISA). The secretion of vascu-lar endothelial cell growth factor (VEGF) and hypoxia inducible factor-1α (HIF-1α) The effect of FIM-A on mRNA and protein expression of mTOR, p70S6 kinase and 4E-1, 4E-BP1 in MG-63 cells was detected by Western blot-ting . Results: Compared with human osteoblast hF-OB1.19 cells, the expression of mTOR, p70s6k and 4E-BP1 mRNA in human osteosarcoma MG-63 cells was significantly increased (P <0.05). FIM-A inhibited the proliferation of MG-63 cells (P <0.05) in a dose-dependent manner (r = 0.940, P <0.01). Compared with the control group, the proportion of MG-63 cells in G0 / G1 phase increased significantly ([(56.4 ± 3.2) vs (43.4 ± 6.9)%, P <0.05] after treatment with 1 × 10-6 mol / L FIM- , While the apoptosis rate of MG-63 cells did not change significantly. The expression of HIF-1α and VEGF in MG-63 cells was significantly lower than that of the control group (P <0.05) after treated with different concentrations of FIM-A for 24 h, with a dose-dependent manner (HIF-1α, r = 0.01; VEGF, r = -0.998, P <0.01). Meanwhile, FIM-A inhibited phosphorylation of mTOR (r = -0.919, P <0.01), p70s6k (r = -0.843, P <0.01) and 4EBP1 (r = -0.818, Also has a concentration-dependent inhibition. Conclusion: FIM-A can inhibit the proliferation of human osteosarcoma MG-63 cells and arrest the cell cycle in G0 / G1 phase. The mechanism may be related to the phosphorylation of mTOR signaling pathway.