活性氧介导的铁过载对小鼠骨髓间充质干细胞的影响及其机制探讨  

Effect of reactive oxygen species mediated iron overload on murine bone marrow mesenchymal stem cells and its mechanism

作　　者：沈继春[1] 张宇辰[2] 赵明峰[2] 

机构地区：[1]武警后勤学院附属医院血液科,天津300162 [2]天津医科大学第一中心临床学院血液科,300192

出　　处：《解放军医学杂志》2015年第2期97-103,共7页Medical Journal of Chinese People's Liberation Army

基　　金：国家自然科学基金(81041043);天津市自然科学基金(13JCYBJC23400);天津市卫生局科技基金(2013KR07)~~

摘　　要：目的建立小鼠骨髓间充质干细胞(BM-MSCs)铁过载(IO)模型,并对铁过载模型小鼠进行去铁及抗氧化治疗,探讨铁过载对小鼠BM-MSCs的损伤作用及活性氧(ROS)在该损伤中的作用机制。方法采用随机区组设计,将40只雄性C57BL/6小鼠随机分为对照组、铁剂(右旋糖酐铁,25mg/ml)组(IO组)、铁剂+去铁治疗(DFX,125mg/kg)组(Fe+DFX组)、铁剂+抗氧化治疗(NAC,40mmol/L)组(Fe+NAC组),每组10只。从小鼠密质骨中分离BM-MSCs培养至P1代,检测BM-MSCs内铁颗粒、不稳定铁(LIP)及ROS水平;利用倍增时间及CCK-8试剂盒检测BM-MSCs增殖情况;采用碱性磷酸酶染色(ALP)、茜素红染色、成骨分化基因检测等方法评估BM-MSCs成骨分化能力;采用油红O染色检测BM-MSCs成脂定向分化能力。结果与对照组相比,铁剂组BM-MSCs内存在明显铁颗粒,LIP及ROS水平明显增高(P<0.05),倍增时间明显延长(2.07±0.14d vs 1.03±0.07d,P<0.01)。DFX组及NAC组倍增时间较铁剂组有所缩短,分别为1.52±0.07d与1.68±0.03d(P<0.05)。与对照组比较,铁剂组BM-MSCs矿化能力及向成骨细胞分化能力下降,成骨基因ALP、RUNX2、OSN表达增强,而成脂定向分化能力增强。在去铁及抗氧化治疗后,上述改变发生部分逆转。结论铁过载可影响小鼠骨髓MSCs的增殖及定向分化能力,其机制可能与铁过载所致ROS升高有关。Objective To reproduce an iron overload （IO） model of murine bone marrow derived mesenchymal stem cells （BM-MSCs）, explore the effects of IO on murine BM-MSCs, and elucidate the involvement of reactive oxygen species （ROS） in this process. Methods Forty male mice （CS7BL/6） were randomly divided into 4 groups （n=10）： control group, IO group, Fe＋iron-chelation （DFX, 125mg/kg） group and Fe＋anti-oxidation （NAC, 40mmol/L） group. BM-MSCs were isolated from compact bone. The levels of iron particles, labile iron pool （LIP） and ROS in BM-MSCs were measured to confirm oxidative stress in the model. Cell proliferation was measured through population double time （DT） and by Cell Counting Kit-8（CCK-8） assay. The osteoblastic differentiation ability of BM-MSCs was assessed by alkaline phosphatase （ALP） activity, alizarin red staining and osteogenic differential genes assay. The adipogenic differentiation ability of BM-MSCs was detected by Oil-Red-O staining. Results Compared with control group, iron deposite increased significantly with higher levels of LIP and ROS in BM-MSCs of IO group （P〈0.05）. In IO group, the BM-MSCs showed a longer double time than that in control group （2.07± 0.14d vs 1.03 ± 0.07d）, which can be reversed to 1.52 ± 0.07d by DFX or to 1.68 ± 0.03d by NAC （P〈0.05）. IO inhibited osteogenic differentiation and mineralization of BM-MSCs, which could be attributed to decreased expression of osteogenic gene alkaline phosphatase （ALP）, runt-related transcription factor 2 （RUNX2） and osteocalcin （OSN）. The osteoblastic differentiation ability of BM-MSCs in IO group was suppressed by IO-induced ROS upregulation. NAC or DFX treatment could partially attenuate cell injury and inhibit the signaling pathway induced by excessive iron. Conclusion I0 may impair the proliferation and differetiation ability of murine BM-MSCs by enhancing the generation of ROS.

关 键 词：铁超负荷 间质干细胞 活性氧 

分 类 号：R551.3[医药卫生—血液循环系统疾病]