摘要
[目的]探讨Notch1 siRNA对乏氧环境人骨肉瘤细胞MG-63细胞增殖和周期的影响。[方法]将骨肉瘤细胞株MG-63细胞置于乏氧(37℃、1%O2、5%CO2)环境下培养作为实验组,以正常氧环境(37℃、21%O2、5%CO2)下培养作为对照组,培养72 h,应用MTT方法检测不同时间乏氧培养对MG-63细胞增殖的影响;采用Notch siRNA转染乏氧环境中的MG-63细胞,MTT方法检测细胞增殖变化;流式细胞术检测细胞周期的变化;Western blot检测细胞HIF-1a、Notch1蛋白表达变化。[结果]乏氧培养可促进MG-63细胞增殖,48 h常氧及乏氧对细胞增殖的作用可见明显差异(2.164±0.075 vs 1.421±0.086)(P<0.01)。流式细胞分析显示实验组、对照组MG-63细胞培养48 h G1/G2期细胞比例分别为(41.79±3.25)%和(53.87±2.31)%(P<0.05),乏氧可以促进细胞周期进展,Notch1 siRNA有效下调乏氧MG-63细胞Notch1蛋白表达,Notchl siRNA作用48 h后乏氧环境MG-63细胞周期阻滞于G1/G2期。Western-blot结果显示乏氧环境下MG-63细胞HIF-1a、Notch1蛋白表达增高。[结论]乏氧环境能通过促进MG-63细胞周期进展,从而明显促进细胞增殖,阻断Notch可逆转乏氧对MG-63细胞促增殖作用,提示Notchl是治疗骨肉瘤的有效分子靶点。
[Objective] We sought to investigate the effects of Notch1 siRNA on the cell cycle and proliferation of the osteosarcoma cell line MG- 63 under hypoxic conditions.[Method]MG- 63 cells were divided in two groups： a hypoxia group（ 37°C,1% O2,5% CO2） and normoxia group（ 37 °C,21% O2,5% CO2）. After 72 h of cultivation,we assessed the effect of hypoxia on MG- 63 cell proliferation at different time points was assessed by MTT assay. MG- 63 cells were transfected with Notch1 siRNA to inhibit its expression,then the cells were subjected to the hypoxic environment. Cell proliferation at different time points was assessed by the MTT assay. Cell cycle regulation was examined using flow cytometry. HIF- 1a and Notch1 protein levels were analyzed by western blot.[Result]As assessed by the MTT assay,the optical density of the hypoxia vs. normoxia group after 48 h of cultivation was 2. 164 ± 0. 075 vs. 1. 421 ± 0. 086,respectively（ P〈0. 01）. Hypoxia promoted MG- 63 cell proliferation,which was obvious at 48 h（ P〈0. 01）. The proportion of cells in the G0 /G1 phase in the hypoxia and normoxia groups after 48 h in culture was assessed by flow cytometry. The proportions of cells in G0 /G1 in the hypoxia vs. normoxia group were 41. 79% ± 3. 25% vs. 53. 87% ± 2. 31%,respectively,thus demonstrating that hypoxia decreased the proportion of cells in the G0 /G1 phase（ P〈0. 05）. Furthermore,the expression of the HIF- 1a and Notch1 proteins was noticeably increased in the hypoxia group. Notch1 siRNA downregulated its protein expression,which inhibited cell proliferation by inducing G0 /G1 phase arrest in MG- 63 cells under hypoxic conditions. [Conclusion]Hypoxia promotes MG- 63 cell proliferation by decreasing the proportion of cells in the G0 /G1 phase. This phenomenon can be reversed by siRNA- mediated knock down of Notch1 expression,thus indicating that Notch1 may be an effective target for the treatment of osteosarcoma.
出处
《中国矫形外科杂志》
CAS
CSCD
北大核心
2014年第18期1691-1695,共5页
Orthopedic Journal of China
基金
山东省自然科学基金(编号:ZR2012HL38
2009ZRA09006)
山东省优秀中青年科学家科研奖励基金(编号:BS2013SF030)
山东省医药卫生科技发展计划项目(编号:2011HW077)资助
