体外磁场定向迁移的间充质干细胞对脊髓损伤的治疗和GAP-43的表达

Effects of target delivery of bone marrow mesenchymal stem cells driven by magnetic field on a rat model of spinal cord injury and GAP-43 expression

目的探讨体外磁场定向迁移尾静脉注射的SPIO标记的大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)对大鼠脊髓损伤治疗的影响以及对脊髓中生长相关蛋白-43(growth-associated protein,GAP-43)表达的影响。方法体外培养SD大鼠BMSCs,流式细胞仪检测其免疫表型,超顺磁氧化铁颗粒(superparamagetic iron oxieds,SPIO)标记BMSCs,普鲁士蓝染色测定SPIO标记率,台盼蓝染色检测标记细胞存活率,CCK-8检测标记细胞分裂增殖能力,Western blot检测标记细胞干细胞标记物Nestin及GFAP表达。建立大鼠脊髓损伤(spinal cord injury,SCI)模型,尾静脉注射移植细胞,对细胞移植后大鼠进行BBB评分。普鲁士蓝染色观察脊髓组织内标记细胞,TTC染色观察脊髓缺血损伤面积,Western blot检测损伤脊髓内GAP-43表达情况。结果原代BMSCs体外能成功分离、培养、传代,且流式细胞仪检测表达间充质干细胞标记物不表达造血干细胞标记物。普鲁士蓝染色显示SPIO标记率接近100%。台盼蓝染色显示标记细胞生存率与对照组无差异(P>0.05)。CCK-8检测磁场下标记细胞增殖活性不受影响(P>0.05)。Western blot检测磁场下标记细胞表达Nestin及GFAP无差异,分化活性不受影响(P>0.05)。磁铁组大鼠BBB评分明显优于对照组(P<0.05)。普鲁士蓝染色观察磁铁组脊髓组织中BMSCs明显多于无磁铁组。TTC染色显示磁铁组缺血面积小于无磁铁组(P<0.05)。Western blot检测脊髓组织中同一时间点GAP-43表达,磁场组明显大于对照组及无磁场组(P<0.05)。结论外置磁场可以定向更多SPIO标记的BMSCs到达脊髓损伤处,使其更好地发挥修复作用且上调GAP-43的表达,利于脊髓损伤的恢复。

Objective To investigate the effects of target delivery of superparamagnetic iron oxide （SPIO）-labeled bone marrow mesenchymal stem cells （BMSCs） driven by magnetic field in vitro on a rat model of spinal cord injury （SCI） and on growth-associated protein 43 （GAP-43） expression in spinal cord.Methods BMSCs from Sprague-Dawley （SD） rats were cultured in vitro. The expression of CD29, CD45, and CD54 was checked by flow cytometry. The BMSCs were labeled with SPIO with label rate tested by Prussian blue staining. The viability of the labeled cells was evaluated by trypan blue staining. CCK-8 assay was used to detect the proliferation of the labeled cells in the presence of magnetic field. Effects of magnetic field on the differentiation of labeled cells were investigated by Western blotting for detection of nestin and GFAP expression. Rats were induced by the clip compression method to establish the SCI model. The BMSCs labeled with SPIO were injected in tail vein. Neurological functions were evaluated using Basso-Beattie-Bresnahan （BBB） score in 3 and 7 d after cells transplantation. The transplanted labeled cells in recipient rat spinal cord were analyzed by Prussian blue staining. The spinal cord ischemia area was checked by TTC staining. The GAP-43 protein expression in the spinal cord of SCI rats after cell transplantation was investigated by Western blotting. Results Flow cytometry results showed that BMSCs expressed the mesenchymal stem cells markers CD29 and CD54 but not the hematopoietic stem cells marker CD45. The Prussian blue staining results showed that the percentage of BMSCs labeled with SPIO was close to 100%. Trypan blue staining showed that there was no significant difference on the viability between the labeled cells and the control group （P〉0.05）. CCK-8 assay did not show any significant difference on proliferation between the magnetic field-treated group and the control group （P〉0.05）. The Western blot results showed that the presence of magnetic field had no effect on the differentiation of BMSCs （P〉0.05）. The BBB score of the magnetic field-treated group was better than that of the control group （P〈0.05）. Prussian blue staining of spinal cord slices showed a higher number of Prussian blue-positive BMSCs in the group with SCI and the presence of magnetic field than that in the SCI group without magnetic field. TTC staining showed the average ischemia size in the spinal cord with magnetic field treatment was significantly smaller than that without magnetic field treatment （P〈0.05）. The Western blot results showed significantly higher expression of GAP-43 in the group with magnetic field treatment as compared to the other two groups on day 3 and day 7 after cell transplantation （P〈0.05）. Conclusion External magnetic field drives the target delivery of more SPIO-labeled BMSCs to the injury area of spinal cord in vivo, relieving SCI and upregulating GAP-43 expression which helps the recovery of SCI.