辛伐他汀动员干细胞归巢对大鼠脊髓损伤的修复作用及其机制

Study on the therapeutic effect of simvastatin by mobilizing the bone marrow-derived stem cells to the injury site in spinal cord injury rat

中文关键词:  脊髓损伤  辛伐他汀  骨髓间充质干细胞  归巢  大鼠

英文关键词:Spinal cord injury  Simvastatin  Bone marrow-derived stem cells  Homing  Rat

中文摘要:

  【摘要】 目的：观察辛伐他汀动员骨髓间充质干细胞（bone marrow stromal cells，BMSCs）归巢至脊髓损伤部位对损伤脊髓的修复作用，并探讨其相关机制。方法：成年雌性SD大鼠30只，经尾静脉注射移植大鼠转绿色荧光蛋白（green fluorescence protein，GFP）基因的BMSCs，24h后随机分为3组：假手术组，仅切除椎板，不损伤脊髓；对照组，脊髓损伤后经蛛网膜下腔注射载体；实验组，脊髓损伤后经蛛网膜下腔注射辛伐他汀。每组10只。对照组和实验组采用改良Allen法（40g·cm）制作T9~T10节段脊髓损伤模型，从L4水平蛛网膜下腔注射辛伐他汀（5mg/kg）或载体。术前及术后1d、3d、7d、14d、21d、28d时盲法进行BBB评分和斜板试验；术后28d处死大鼠，取相应节段脊髓组织行形态计量学观察脊髓空腔体积大小；应用神经元核抗原（NeuN）/神经胶质纤维酸性蛋白（GFAP）与GFP免疫荧光双染法观察移植GFP-BMSCs的迁移及细胞分化情况；Western blot检测血管内皮生长因子（vascular endothelial growth factor，VEGF）及脑源性神经营养因子（brain derived neurotrophic factor, BDNF）表达水平的变化。结果：术后各时间点假手术组动物BBB评分及斜板试验角度无明显变化，实验组及对照组BBB评分及斜板试验角度明显降低，术后7d、14d、21d、28d时实验组BBB评分高于对照组（P＜0.05）；术后14d、21d、28d时实验组大鼠的斜板试验角度高于对照组（P＜0.05）。术后28d时假手术组脊髓内未见空腔，实验组和对照脊髓损伤部位均可见空洞形成，实验组空腔体积（0.29±0.08mm3）小于对照组（0.57±0.10mm3）（P＜0.05）；荧光显微镜下观察实验组在脊髓损伤周边可见大量表达绿色荧光的细胞，而对照组和假手术组很少。免疫荧光染色实验组NeuN（+）/GFAP（+）、GFP（+）/GFAP（+）细胞明显多于对照组和假手术组（P＜0.05）。Western blot显示治疗组大鼠损伤处脊髓BDNF和VEGF表达高于对照组和假手术组（P＜0.05）。结论：辛伐他汀可动员BMSCs归巢至脊髓损伤部位并参与损伤修复，其作用可能与其促进BDNF及VEGF高表达有关。

英文摘要:

  【Abstract】 Objectives: To study the effect of simvastatin-mediated mobilization of bone marrow-derived stem cells(BMSCs) on the injured spinal cord and the underlying mechanism. Methods: The transgenic rat GFP-BMSCs were transplanted into rat through the caudal vein. 24 hours after transplantation, animals were randomly divided into 3 groups: sham group(laminectomy only), vehicle treated group(SCI+vehicle) and simvastatin treated group(SCI+simvastatin). Spinal cord injury was introduced by modified Allen′s method (10g·4cm) at T10 level. Simvastatin or vehicle was injected into the subarachnoid space after injury. Hind limb locomotor recovery(BBB scores and inclined plane test) was used for assessment. At 28 days after injury, animals were killed and HE was performed to evaluate the area of spinal cavity. Immunohistochemistry for GFP and cell lineage marker NeuN and GFAP was performed to evaluate simvastatin-mediated mobilization of bone marrow-derived cells into injured spinal cord. Western blot was performed to detect the expression of BDNF and VEGF. Results: The BBB scores and inclined-plated angles of the sham group did not significanlty change during the research. While those in simvastatin treated rats showed significant recovery of hind limb function compared to control rats. HE revealed that simvastatin decreased the cavity of injured spinal cord. Immunohistochemistry revealed that simvastatin increased the number of GFP-positive cells in injured spinal cord, indicating that bone marrow-derived cells were mobilized and migrated into injured spinal cord. The numbers of double positive cells for GFP and NeuN marker or GFAP were more in the simvastatin treated rats than in the control rats, indicating that these GFP-BMSCs differentiated into the neuro or glial cells. The expression levels of BDNF and VEGF were higher in the simvastatin treated group. Conclusions: Simvastatin can mobilize the BMSCs home to the injured spinal cord, which can decrease the spinal cavity, increase the expression of BDNF and VEGF, and improve the functional recovery of spinal cord injury.