摘要
目的探讨铁过载对脐带间充质干细胞（UC-MSC）的损伤作用及活性氧物质（ROS）在该机制中的意义。方法体外培养UC．MSC，用不同浓度的枸橼酸铁胺（FAC）处理UC-MSC不同时间建立铁过载模型。采用细胞倍增时间计算细胞的增殖能力；用AnnexinV／PI双染法检测细胞的凋亡；用共培养方法检测UC-MSC的造血支持能力；并用DCFH-DA荧光探针检测细胞内ROS水平；Western印迹法检测铁过载及抗氧化处理后UC-MSC内ROS相关信号蛋白p-p38MAPK、p38MAPK及1953的表达。结果（1）加铁组UC-MSC的群体倍增时间明显长于对照组[第3代细胞：（24．43±2．72）h比（16．03±2．31）h，P〈0．05]，但传代次数增加2代后两组之间差异无统计学意义（P〉0．05）；（2）加铁组UC-MSC的凋亡率（12．75％±0．32％）明显高于对照组（3．63％±0．80％）（P〈0．05）；（3）脐血单个核细胞（MNC）与加铁组UC-MSC共培养1和2周时，其造血集落形成能力均明显低于对照组（均P〈0．05）；（4）加铁组UC-MSC内ROS水平明显高于对照组，且ROS的水平呈时间与浓度依赖性，在400LLm0L／LFAC浓度下作用12h达到最高值（1499±86比548±97，P〈0．05）；（5）加铁组UC-MSC的p-p38MAPK、P53蛋白表达明显高于对照组，当给予抗氧化剂处理后，相关信号通路可被抑制。结论铁过载可能通过激活ROS相关信号传导因子p-p38MAPK、1953抑制UC-MSC的增殖能力，诱导其凋亡，降低其造血支持能力。
Objective 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. Methods 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 diaeetate （DCFH-DA）. And the ROS related signaling factors of p-p38MAPK, p38 MAPK, P53 were measured by Western blot. Results （ 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. Conclusions Ironoverload may impair the proliferation, survival and hematopoeisis supportive function of UC-MSC by enhancing the generation of ROS. And ROS stimulates the signaling pathways of p-p38MAPK and P53.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2013年第12期930-934,共5页
National Medical Journal of China
基金
国家自然科学基金（81041043）
教育部留学归国人员科研启动基金（教外司留[2007]1108）
天津市卫生局科技基金重点项目（2011KR01）
关键词
铁超负荷
间质干细胞
脐带
活性氧物质
信号传导
Iron overload
Mesenchymal stem-cells
Umbilical cord
Reactive oxygenspecies
Signal transduction
