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目的:探讨缺氧对骨肉瘤细胞MG63核干细胞因子(NS)表达的调节及其机制。方法:反转录聚合酶链式反应(RT-PCR)和蛋白质印迹法检测缺氧对NS转录和蛋白表达的影响;蛋白质印迹法检测在雷帕霉素抑制缺氧诱导因子-1α(HIF-1α)表达或激酶抑制剂预处理后NS在缺氧下的变化,检测NS调节的下游因子p53和活性Caspase-3蛋白表达的变化。结果:缺氧6、12和24 h后NS蛋白表达分别升高了1.26(P>0.05)、1.58(P<0.05)和1.92倍(P<0.01),NS的转录分别升高了1.33(P>0.05)、1.49(P<0.05)和1.54倍(P<0.05)。蛋白激酶B(Akt)抑制剂wortmannin逆转了缺氧对NS的上调,而细胞外信号调节激酶(ERK)抑制剂U0126、蛋白激酶A(PKA)抑制剂H89和雷帕霉素则没有逆转缺氧对NS的上调。Akt活性在缺氧6、12和24 h后分别升高了1.51(P>0.05)、1.56(P<0.05)和1.23倍(P>0.05)。尽管缺氧上调了NS的表达,但NS的下游因子p53与活性Caspase-3蛋白表达在缺氧后无明显变化。结论:缺氧通过增强Akt的活性上调了NS的表达。
Objective: To investigate the regulation of hypoxia on the expression of nuclear factor of nuclear sclerotiorin (MG63) in osteosarcoma cell line MG63 and its mechanism. Methods: Reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting were used to detect the effect of hypoxia on NS transcription and protein expression. Western blotting was used to detect the effect of rapamycin on hypoxia inducible factor-1α (HIF- 1α) expression or kinase inhibitor pretreatment of NS in hypoxia changes, detection of NS regulation of downstream factors p53 and active caspase-3 protein expression changes. Results: The expressions of NS protein increased by 1.26 (P> 0.05), 1.58 (P <0.05) and 1.92 times (P <0.01) > 0.05), 1.49 (P <0.05) and 1.54-fold (P <0.05). Wortmannin, an inhibitor of protein kinase B (Akt), reversed the upregulation of NS by hypoxia, whereas extracellular signal-regulated kinase (ERK) inhibitor U0126, protein kinase A (PKA) inhibitor H89 and rapamycin did not reverse hypoxia The increase of NS. Akt activity increased by 1.51 (P> 0.05), 1.56 (P <0.05) and 1.23 times (P> 0.05) after 6, 12 and 24 h of hypoxia, respectively. Although hypoxia up-regulated the expression of NS, the expression of p53 and Caspase-3, a downstream factor of NS, did not change significantly after hypoxia. Conclusion: Hypoxia up-regulates NS expression by increasing Akt activity.