缺氧对骨肉瘤细胞系MG-63 HIF-1α、p53 、bcl-2 及细胞凋亡的影响

 目的 了解缺氧环境对骨肉瘤MG-63细胞缺氧诱导因子HIF-1α、p53、bcl-2的表达及细胞凋亡的影响。方法 建立骨肉瘤细胞体外缺氧模型,观察不同缺氧时间段（8、16、24h）HIF-1α、p53、bcl-2的表达和细胞凋亡的情况。半定量RT-PCR方法检测HIF-1α、p53、bcl-2的表达水平;免疫组化（SP法）和免疫印迹（WesternBlot）检测HIF-1α、p53、bcl-2蛋白表达情况;流式细胞仪检测细胞凋亡率。结果 缺氧处理后,HIF-1α转录水平未见明显改变,蛋白表达水平随缺氧时间延长明显增强;而p53、bcl-2mR-NA及蛋白表达水平均显著增强,两者间存在一定的相关性;细胞凋亡率却未见明显增加。结论 在缺氧条件下,不能通过以HIF-1α为中介的p53依赖途径来诱导骨肉瘤MG-63细胞的凋亡,其机制可能与缺氧诱导野生型p53的突变或缺失使HIF-1α和bcl-2的过表达有关。

Effect of Hypoxia on the Expression of Hypoxia Inducible Factor 1α、p53、bcl-2 and Apoptosis in Osteosarcoma Cell Line MG-63

Objective 　To investigate the effect of hypoxia on the expression of ypoxia inducible factor 1α (HIF-1α) 、p53 、bcl-2 and apoptosis in osteosarcoma cell line MG263 under hypoxia environment . Methods The hypoxia culture model was established by a hypoxia incubator. The gene productions of HIF-1α、p53 、bcl-2 and the changes of the apoptosis were observed at different hypoxia culture phase. The semi-quantitative reverse t ranscriotion PCR(RT-PCR) was used to test the mRNA expression of HIF-1α、p53 and bcl-2. The protein level of HIF-1α、p53 and bcl-2 was observed by immunohistochemical staining and western blot analysis. The changes of apoptosis were analyzed using flow cytometry. Results 　After the hypoxia treatment ,the mRNA level of HIF-1αwas not changed significantly ,however ,the protein expression of HIF-1αwas increased remarkably with correspondence to the hypoxia time. But the expression of p53 and bcl-2 were up-regulated in mRNA and protein level .Besides ,the bcl-2 activity was markedly associated with the level of HIF-1α. However ,the apoptosis rate was not increased markedly. Conclusion 　In hypoxia environment ,the apoptosis of osteosarcoma cell line MG-63 could not induce by the p53 avenue which depended on HIF-1α. The mechanism is possibly related with the mutation of p53 that increases the expression of HIF-1αand bcl-2.