CAJ | 학술논문

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P53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400 μmol/L FAC浓度下作用12 h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、P53抑制UC-MSC的增殖能力,诱导其凋亡,降低其造血支持能力.
목적 탐토철과재대제대간충질간세포(UC-MSC)적손상작용급활성양물질(ROS)재해궤제중적의의.방법 체외배양UC-MSC,용불동농도적구연산철알(FAC)처리UC-MSC불동시간건립철과재모형.채용세포배증시간계산세포적증식능력；용AnnexinV/PI쌍염법검측세포적조망；용공배양방법검측UC-MSC적조혈지지능력；병용DCFH-DA형광탐침검측세포내ROS수평；Western인적법검측철과재급항양화처리후UC-MSC내ROS상관신호단백p-p38MAPK、p38MAPK급P53적표체.결과 (1)가철조UC-MSC적군체배증시간명현장우대조조[제3대세포:(24.43±2.72)h비(16.03±2.31)h,P＜0.05],단전대차수증가2대후량조지간차이무통계학의의(P＞0.05)；(2)가철조UC-MSC적조망솔(12.75％±0.32％)명현고우대조조(3.63％±0.80％)(P＜0.05)；(3)제혈단개핵세포(MNC)여가철조UC-MSC공배양1화2주시,기조혈집락형성능력균명현저우대조조(균P ＜0.05)；(4)가철조UC-MSC내ROS수평명현고우대조조,차ROS적수평정시간여농도의뢰성,재400 μmol/L FAC농도하작용12 h체도최고치(1499±86비548±97,P＜0.05)；(5)가철조UC-MSC적p-p38MAPK、P53단백표체명현고우대조조,당급여항양화제처리후,상관신호통로가피억제.결론 철과재가능통과격활ROS상관신호전도인자p-p38MAPK、P53억제UC-MSC적증식능력,유도기조망,강저기조혈지지능력.
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 diacetate (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 Iron overload 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.