铁蓄积诱导骨髓间充质干细胞凋亡并抑制成骨活性的研究

Iron accumulation induces bone marrow mesenchymal stem cells apoptosis via cysteinyl - aspartate -specific proteases- 3 activation and inhibits osteogenesis

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）0.1 g/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（P1NP），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24 h，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。

Objective 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）, Procol-lagen type 1 N -terminal propeptide, osteogenesis markers （P1NP）, distal femur trabecular 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 cultivat- ed 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 con- trol 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/nd 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） rag/ram3 （ 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） mini decreased significantly compared with control group [ BV/TV ： 19.92% ± 1.92% , Tb. Th ： （ 0.098 ± 0. 008 ） mm, Tb. Sp ： （0. 172 ± O. 133 ） mm, P=0. 021]. Conclusion The osteogenetic activity is re- strained after iron accumulation may be associated with increased BMSCs apoptosis, BMSCs apoptosis in- creased may be related to iron accumulation induced Casoase -3 activation.