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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。
Objective To investigate the effects of hypoxic environment on the expression of hypoxia-inducible factor-1 (HIF-1α), p53, bcl-2 and apoptosis in osteosarcoma MG-63 cells. Methods The in vitro hypoxia model of osteosarcoma cells was established to observe the expression of HIF-1α, p53, bcl-2 and apoptosis in different time of hypoxia (8, 16, 24 h). The expression of HIF-1α, p53 and bcl-2 were detected by semi-quantitative RT-PCR. The expressions of HIF-1α, p53 and bcl-2 were detected by immunohistochemistry and Western blotting. Instrument detection of apoptosis rate. Results After hypoxia treatment, there was no significant change in the transcription level of HIF-1α, but the expression of p53 protein, bcl-2mR-NA protein and protein were significantly increased with the prolongation of hypoxia, and there was a certain correlation between them Sex; apoptosis rate but no significant increase. Conclusions Under hypoxia, apoptosis of osteosarcoma MG-63 cells can not be induced by HIF-1α-mediated p53-dependent pathway. The mechanism may be related to hypoxia-induced wild-type p53 mutation or loss of HIF-1α and bcl-2 over-expression.