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目的:通过小分子化合物氯化钴(CoCl2)模拟的低氧环境,分析低氧下及其雷帕霉素(RPM)作用下人急性髓细胞白血病细胞HL-60的低氧调控信号分子表达的变化;方法:常规方法复苏、传代、培养HL-60细胞,培养细胞进入对数生长期后用于实验。低氧模拟组、低氧雷帕霉素处理组、常氧雷帕霉素处理组分别用含200μmol/L CoCl2、200μmol/L CoCl2/20nmol/L RPM、20nmol/LRPM的1640培养基处理生长状态良好的细胞,对照组细胞用1640培养基培养,各组置培养箱以37℃、5%CO2培养,并于处理后24h、48h、72h收集细胞用于检测;采用实时荧光定量PCR方法检测低氧诱导因子(HIF-1α)、内皮细胞生长因子(VEGF)、雷帕霉素靶蛋白(mTOR)及GAPDH在转录水平的表达;结果:①.与各时段对照组相比,低氧模拟组HIF-1α表达随时间逐渐增加,72h明显上调;与常氧雷帕霉素处理组各时段比较,低氧雷帕霉素处理组HIF-1α表达早期(24h)相对下调,后期相对上调;②.与对照组比较,各处理组mTOR表达均下调,低氧雷帕霉素处理组在早期(24h)下调显著;与常氧雷帕霉素处理组比较,低氧雷帕霉素处理组mTOR各时段的表达均相对下调;③与对照组各时段相比,低氧模拟组VEGF的表达在早期显著上调,但后期呈下调;常氧雷帕霉素处理组各时段VEGF的表达下调,与其比较,低氧雷帕霉素处理组各时段均呈相对下调。结论:常氧和低氧下RPM作用HL-60细胞后VEGF、mTOR的mRNA均表达下调,RPM可在低氧环境下增强了这种下调表达作用。
OBJECTIVE: To analyze the expression of hypoxia-regulated signal molecules of human acute myeloid leukemia HL-60 cells under hypoxia and rapamycin (RPM) through hypoxia environment simulated by small molecule cobalt chloride (CoCl2) Methods: HL-60 cells were resuscitated, passaged and cultured in the routine method. The cultured cells were used in the logarithmic growth phase for the experiment. The hypoxia model group, the hypoxia rapamycin group and the noraprenamycin treatment group were respectively treated with 1640 medium containing 200 μmol / L CoCl 2, 200 μmol / L CoCl 2/20 nmol / L RPM and 20 nmol / L RPM for growth state The cells in the control group were cultured in 1640 medium. The cells in each group were cultured at 37 ℃ and 5% CO 2, and the cells were harvested at 24h, 48h and 72h after treatment. Real-time fluorescence quantitative PCR The expression of HIF-1α, VEGF, mTOR and GAPDH at the transcriptional level were analyzed by Western blotting.Results: ① Compared with the control group at each time point, hypoxia model group The expression of HIF-1α gradually increased over time, and was significantly up-regulated at 72h. HIF-1α expression was down-regulated in the early stage of hypoxia-rapamycin treatment compared with that in the normoxic rapamycin-treated group .Compared with the control group, the expression of mTOR was down-regulated in all treatment groups, and down-regulated in hypoxia-rapamycin group at early stage (24h). Compared with the normoxia-rapamycin-treated group, mTOR Compared with the control group, the expression of VEGF in each time period was lower than that in the control group Early significantly upregulated, down-regulated but was late; normoxic expression rapamycin treated group each time the decrease of VEGF and its comparison, hypoxia rapamycin treatment groups showed relatively lowered each time. CONCLUSION: Both mRNA expression of mTOR and VEGF are down-regulated in RPM-60 and HL-60 cells under normoxia and hypoxia, and RPM can enhance this down-regulation in hypoxia.