Lu Wen-yi, Zhao Ming-feng, Chai Xiao, Meng Juan-xia, Zhao Nan, Rajbhandary Sajin, Xu Xin-nü, Ma Li, Li Yu-ming
Department of Hematology & Oncology, Tianjin Medical University, Tianjin, China.
Zhonghua Yi Xue Za Zhi. 2013 Mar 26;93(12):930-4.
To explore the effects of iron overload on umbilical cord derived mesenchymal stem cells (UC-MSC) and elucidate the involvement of reactive oxygen species (ROS) in this process.
The iron overload model of MSC was established by in vitro addition of ferric ammonium citrate (FAC) into culture medium. Cell proliferation and apoptosis were determined by Annexin V/PI double staining and population doubling time (DT) respectively. Co-culture system was used to assess the hematopoietic support capacity of UC-MSC in different groups. Thereafter the ROS level was detected with fluorescent probe 2', 7'-dichlorofluorescin diacetate (DCFH-DA). And the ROS related signaling factors of p-p38MAPK, p38 MAPK, P53 were measured by Western blot.
(1) The DT of UC-MSC in iron overload group was significantly longer than that of control ((24.43 ± 2.72) h vs (16.03 ± 2.31) h, P < 0.05). But the difference was insignificant after two passages (P > 0.05). (2) Apoptosis in iron overload group was higher than that of control (12.75% ± 0.32% vs 3.63% ± 0.80%, P < 0.05). (3) The colony forming capacity of mononuclear cell (MNC) co-cultured with UC-MSC of iron overload group for 1/2 weeks significantly decreased. (4) The ROS level of UC-MSC with iron overload was higher than that of control in time and concentration-dependent fashions and it peaked at 400 µmol/L of FAC for 12 h (1499 ± 86 vs 548 ± 97, P < 0.05). (5) The expressions of p-p38MAPK and P53 increased in response to FAC compared with control. But such an effect was partially inhibited after the use of antioxidants.
Iron overload may impair the proliferation, survival and hematopoiesis supportive function of UC-MSC by enhancing the generation of ROS. And ROS stimulates the signaling pathways of p-p38MAPK and P53.
探讨铁过载对脐带间充质干细胞（UC-MSC）的影响，并阐明活性氧（ROS）在这一过程中的作用。
通过在培养基中体外添加柠檬酸铁铵（FAC）建立MSC的铁过载模型。分别采用Annexin V/PI双染法和群体倍增时间（DT）测定细胞增殖和凋亡。采用共培养系统评估不同组UC-MSC的造血支持能力。此后，用荧光探针2',7'-二氯荧光素二乙酸酯（DCFH-DA）检测ROS水平。并用蛋白质免疫印迹法检测ROS相关信号因子p-p38MAPK、p38 MAPK、P53。
（1）铁过载组UC-MSC的DT显著长于对照组（（24.43±2.72）小时对（16.03±2.31）小时，P<0.05）。但传代两次后差异无统计学意义（P>0.05）。（2）铁过载组的凋亡率高于对照组（12.75%±0.32%对3.63%±0.80%，P<0.05）。（3）与铁过载组UC-MSC共培养1/2周的单核细胞（MNC）集落形成能力显著降低。（4）铁过载的UC-MSC的ROS水平在时间和浓度依赖性方式上高于对照组，并在400µmol/L FAC处理12小时时达到峰值（1499±86对548±97，P<0.05）。（5）与对照组相比，FAC处理后p-p38MAPK和P53的表达增加。但使用抗氧化剂后这种作用部分受到抑制。
铁过载可能通过增强ROS的产生损害UC-MSC的增殖、存活和造血支持功能。并且ROS刺激p-p38MAPK和P53信号通路。
