摘要目的 观察铁蓄积对骨髓间充质干细胞(BMSCs)数量、凋亡的影响,探讨铁蓄积在骨质疏松BMSCs层面的发病机制.方法 体内实验,6～8周C57雄性小鼠分为对照组、实验组,实验组用枸橼酸铁铵(FAC)0.1g/kg干预8周.使用酶联免疫吸附试验(ELISA)检测血清铁蛋白(FER)、成骨标记物Ⅰ型前胶原氨基末端肽(P1 NP),micro-CT进行股骨远端骨小梁三维形态重建和空间结构参数分析,流式细胞仪检测BMSCs占骨髓细胞比例.细胞实验,取6只8周C57雄性小鼠骨髓组织,体外实验分离培养BMSCs,达到一定数量后分为细胞对照组和细胞实验组,实验组加FAC干预24h,使用流式细胞仪检测细胞凋亡,Western blot检测天冬半胱氨酸特异性蛋白酶(Caspase-3)表达水平.收集在体观察、细胞观察各组实验数据统计分析.结果 FAC组BMSCs占骨髓比例[(2.68±0.91)％]与对照组[(4.06±0.76)％]比较显著降低(P=0.029)、FAC组BMSCs细胞凋亡比例[(1.86±0.22)％]比较于对照组[(0.25±0.09)％]显著增加(P=0.032)、BMSCs细胞Caspase-3蛋白切割增加;ELISA结果显示FAC组成骨指标P1NP(4.08±0.71) ng/ml比较于对照组(9.16±0.55) ng/ml显著降低(P=0.041)、FAC组血清铁蛋白(14.34±0.82)比较于对照组(5.89±0.62)显著上升(P=0.021),micro-CT检测的FAC组骨密度(50.41±10.27) mg/mm3比对照组(104.68±8.56) mg/mm3显著下降(P =0.033)、FAC组骨小梁空间结构参数[BV/TV骨体积分数:(10.18±1.76)％,Tb.Th骨小梁厚度:(0.057±0.012) mm,Tb.Sp骨小梁分离度:(0.322±0.214) mm]比较于对照组[BV/TV:(19.92±1.92)％,Tb.Th:(0.098±0.008) mm,Tb.Sp:(0.172±0.133) mm]显著下降(P =0.021).结论 铁蓄积后,成骨活性指标受到抑制可能与BMSCs凋亡增加相关,而BMSCs凋亡增加可能与铁蓄积诱导Caspase-3活化相关.

abstractsObjective To study iron accumulation affect on bone marrow mesenchymal stem cells (BMSCs) number and apoptosis,and to explore iron accumulation in iron accumulation osteoporosis BMSCs level pathogenesis.Methods In vivo,6-8 weeks C57 male mice were devived into control group and experimental group,the experimental group was intraperitoneally injected with ferric ammonium citrate (FAC) of 0.1 g/kg intervention in eight weeks,the two groups were tested:serum ferritin (FER),Procollagen type 1 N-terminal propeptide,osteogenesis markers (P1NP),distal femur trabocular bone reconstruction of three dimensional form and spatial structure parameter,BMSCs accounted for the proportion of bone marrow cells.In vitro,6-8 weeks male C57 mice bone marrow tissue were separation,BMSCs were cultivated and devided after reaching a certain number in the control group and experimental group.After 24 h FAC intervention in experimental group,two groups were tested:cell apoptosis,cysteinyl-aspartate-specific proteases-3 (Caspase-3) expression level.Statistical analyses were performed.Results FAC group bone marrow BMSCs [(2.68 ± 0.91) ％] significantly reduced compare with control group [(4.06 ± 0.76) ％](P =0.029),FAC group apoptosis percentage [(1.86 ± 0.22)％] increased compare with control group [(0.25 ±0.09)％,P =0.032],BMSCs Caspase-3 cell protein cutting increased compared with the control group;FER in FAC group (14.34 ±0.82) increased compared with the control group (5.89 ±0.62,P =0.021),FAC group P1NP (4.08 ±0.71) ng/ml osteogenesis index decreased significantly compared with the control group(9.16 ± 0.55) ng/ml (P =0.041),the micro-CT detection of bone mineral density in FAC group (50.41 ± 10.27) mg/mm3 was lower in control group (104.68 ± 8.56) mg/mm3 (P =0.033).FAC group bone trabecular space structure parameters [BV/TV,volume fraction:(10.18 ±1.76)％,Tb.Th,trabecular thickness:(0.057 ± 0.012) mm,Tb.Sp,trabecular space:(0.322 ±0.214) mm]decreased significantly compared with control group [BV/TV:19.92％ ± 1.92％,Tb.Th:(0.098 ±0.008) mm,Tb.Sp:(0.172 ±0.133) mm,P =0.021].Conclusion The osteogenetic activity is restrained after iron accumulation may be associated with increased BMSCs apoptosis,BMSCs apoptosis increased may be related to iron accumulation induced Caspase-3 activation.