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目的探讨铁过载催化的活性氧(ROS)生成对骨髓间充质干细胞(MSCs)增殖和凋亡的影响及其可能的作用机制。方法体外培养MSCs,加入不同浓度(100、200、400μmol/L)的枸橼酸铁铵(FAC),培养12、24、48 h,建立铁过载模型,测定铁过载前后细胞内的不稳定铁池(LIP)和ROS水平,并采用群体倍增时间(DT)检测MSCs的细胞增殖能力,采用Annexin V-PI双标法检测细胞凋亡率,进行蛋白质印迹实验检测磷酸化p38丝裂原活化蛋白激酶(P-p38MAPK)、p38MAPK、蛋白激酶B(AKT)及p53蛋白的表达情况。结果铁过载组MSCs的LIP水平明显高于对照组(平均荧光强度482.49±20.96比303.88±23.37,P<0.05);ROS的水平随FAC浓度增加而升高,在加入FAC浓度为400μmol/L时最高(P<0.05)。用400μmol/L的FAC分别处理12、24、48 h的MSCs DT分别为(1.47±0.11)、(1.80±0.13)、(2.04±0.14)d,均明显长于对照组的(1.20±0.05)d(P均<0.05)。铁过载组MSCs的凋亡率也明显高于对照组[(3.51±1.17)%比(0.66±0.62)%,P<0.05]。与对照组比较,铁过载组P-p38MAPK、p38MAPK、p53蛋白的表达增加,而AKT的表达无明显差异。结论铁过载可通过提高骨髓MSCs的ROS水平来抑制其增殖、诱导其凋亡,此过程可能与p38MAPK-p53信号通路的激活有关。
Objective To investigate the effects of iron overload catalyzed by reactive oxygen species (ROS) on proliferation and apoptosis of bone marrow mesenchymal stem cells (MSCs) and their possible mechanism. Methods MSCs were cultured in vitro. Ferric ammonium citrate (FAC) was added into the culture medium at different concentrations (100, 200 and 400μmol / L) for 12, 24, and 48 hours. The iron overload model was established and the intracellular iron (LIP) and ROS levels. The proliferation of MSCs was detected by population doubling time (DT). The apoptosis rate of MSCs was detected by Annexin V-PI double-labeled method. The phosphorylated p38 mitogen-activated protein (P-p38MAPK), p38MAPK, protein kinase B (AKT) and p53 protein expression. Results The level of LIP in MSCs with iron overload group was significantly higher than that in control group (mean fluorescence intensity 482.49 ± 20.96 vs 303.88 ± 23.37, P <0.05). The level of ROS increased with the increase of FAC concentration. When FAC concentration was 400 μmol / L Highest (P <0.05). The DT of MSCs treated with 400μmol / L FAC for 12, 24 and 48 h were (1.47 ± 0.11), (1.80 ± 0.13) and (2.04 ± 0.14) d, respectively, which were significantly longer than those in control group (1.20 ± 0.05) d (P <0.05). The apoptosis rate of MSCs in iron overload group was also significantly higher than that in control group [(3.51 ± 1.17)% vs (0.66 ± 0.62)%, P <0.05]. Compared with the control group, the expression of P-p38MAPK, p38MAPK and p53 increased in iron overload group, while the expression of AKT had no significant difference. Conclusion Iron overload can inhibit the proliferation and induce the apoptosis of MSCs by increasing the level of ROS, which may be related to the activation of p38 MAPK-p53 signaling pathway.