Objective: To investigate the effect of granulocyte colony-stimulating factor (G-CSF) mobilizing the bone marrow mesenchymal stem cells (BMSCs) homing to the spinal cord injury sites in rats, and to evaluate the feasibility of G-CSF mobilizing the BMSCs home to the injured spinal cord.
Methods: Twenty-four healthy adult female Sprague Dawley rats were injected with 1 mL green fluorescence protein labeled BMSCs (GFP-BMSCs, 1×10 6 cells/mL) into tail vein at 12 hours before operation. They were randomly divided into sham operation group (group A), sham operation+G-CSF group (group B), spinal cord injury group (group C), and spinal cord injury+G-CSF group (group D), with 6 rats in each group. In groups C and D, spinal cord injury model was established by T 10 level spinal cord hemisection. In groups A and B, only laminectomy was performed without injury to the spinal cord. Groups B and D were injected with G-CSF (10 μg/kg·d) at 1 hour after operation for 3 consecutive days, and groups A and C were injected with the same amount of saline. The Basso-Beattie-Bresnahan (BBB) score was used to estimate the neurological function of rats and the expressions of tumor necrosis factor α (TNF-α) and stromal-derived factor 1 (SDF-1) were detected by ELISA method at 1, 3, 7, 14, 21, and 28 days after operation. The spinal cord samples of rats were sacrificed at 28 days after operation for immunohistochemical staining to observe the expression of cytokines, including SDF-1, brain derived neurotrophic factor (BDNF), vascular endothelial growth factor (VEGF), and TNF-α, and immunofluorescence staining to observe GFP-BMSCs positive cells, double-stained fluorescent yellow GFP/neuronal nuclear antigen (NeuN) positive neurons, and GFP/glial fibrillary acidic protein (GFAP) positive neurons. The number of glial cells and apoptosis were detected by TUNEL method.
Results: The BBB score of groups A and B had no significant change at each time point after operation. At 1 day after operation, the BBB score of groups C and D decreased to the lowest level, and then gradually increased. The BBB score of group D was significantly higher than that of group C at all time points except 1 day after operation ( P<0.05). At 3, 7, 14, 21, 28 days after operation, the levels of TNF-α and SDF-1 in groups C and D were significantly higher than those in groups A and B ( P<0.05), but the levels of TNF-α in group D were significantly lower than those in group C at each time point, and the levels of SDF-1 were significantly higher than those in group C ( P<0.05). Immunohistochemical staining showed that the expressions of SDF-1, BDNF, VEGF, and TNF-α in groups C and D were significantly higher than those in groups A and B ( P<0.05); the expressions of SDF-1, BDNF, and VEGF in group D were significantly higher than those in group C, and the expression of TNF-α was significantly lower than that in group C ( P<0.05). Immunofluorescence staining showed that the number of GFP-BMSCs, GFP/NeuN, and GFP/GFAP positive cells in groups C and D were significantly higher than those in groups A and B, and in group D than in group C ( P<0.05). TUNEL assay showed that the number of apoptotic cells in groups C and D was significantly lower than that in groups A and B, and in group D than in group C ( P<0.05).
Conclusion: G-CSF can mobilize BMSCs to the spinal cord injury site and promote repair effect by down-regulating TNF-α to promote the anti-apoptosis function and up-regulating SDF-1, BDNF, VEGF to promote BMSCs migration.
目的: 观察粒细胞集落刺激因子（granulocyte colony stimulating factor，G-CSF）动员 BMSCs 归巢对大鼠脊髓损伤的治疗效果，评估 G-CSF 动员 BMSCs 治疗脊髓损伤的可行性。.
方法: 将 24 只成年健康雌性 SD 大鼠术前 12 h 于鼠尾静脉注射绿色荧光蛋白（green fluorescence protein，GFP）标记的 BMSCs（GFP-BMSCs），并随机分为假手术组（A 组）、假手术+G-CSF 组（B 组）、脊髓损伤组（C 组）、脊髓损伤+G-CSF 组（D 组），每组 6 只。C、D 组采用 T 10 水平脊髓半切法建立脊髓损伤模型，A、B 组仅行椎板切除，不损伤脊髓；术后 1 h B、D 组分别注射 G-CSF（10 μg/kg·d），连续注射 3 d；A、C 组注射等量生理盐水。术后 1、3、7、14、21、28 d 采用 BBB 评分行大鼠双后肢神经功能评估，并采用 ELISA 法检测血清 TNF-α 和基质细胞衍生因子 1（stromal cell-derived factor 1，SDF-1）表达。术后 28 d 处死大鼠取脊髓样品行免疫组织化学染色观察细胞因子 SDF-1、BDNF、VEGF 和 TNF-α 表达，免疫荧光染色观察 GFP-BMSCs 阳性细胞及双染荧光黄色的 GFP/神经元核抗原（neuronal nuclei，NeuN）阳性神经元细胞和 GFP/胶质原纤维酸性蛋白（glial fibrillary acidic protein，GFAP）阳性神经胶质细胞数；并采用 TUNEL 法检测细胞凋亡。.
结果: 术后各时间点 A、B 组 BBB 评分较术前无明显变化；术后 1 d，C、D 组 BBB 评分降至最低，后逐渐上升。除术后 1 d 外，其余各时间点 D 组 BBB 评分均显著高于 C 组（ P<0.05）。术后 3、7、14、21、28 d，C、D 组 TNF-α 和 SDF-1 含量均明显高于 A、B 组（ P<0.05）；但 D 组各时间点 TNF-α 含量显著低于 C 组，SDF-1 含量显著高于 C 组（ P<0.05）。免疫组织化学染色示，术后各时间点 C、D 组 SDF-1、BDNF、VEGF 和 TNF-α 表达均显著高于 A、B 组（ P<0.05）；D 组 SDF-1、BDNF、VEGF 表达显著高于 C 组，TNF-α 表达显著低于 C 组（ P<0.05）。免疫荧光染色示，C、D 组 GFP-BMSCs、GFP/NeuN、GFP/GFAP 阳性细胞数均显著多于 A、B 组，D 组显著多于 C 组，差异均有统计学意义（ P<0.05）。TUNEL 法检测示，C、D 组凋亡细胞数目显著低于 A、B 组，D 组显著低于 C 组，差异均有统计学意义（ P<0.05）。.
结论: G-CSF 可以动员 BMSCs 归巢至大鼠脊髓损伤部位并参与修复，其作用可能与其下调 TNF-α 减少细胞凋亡，上调 SDF-1、BDNF、VEGF 促进 BMSCs 迁移有关。.
Keywords: Spinal cord injury; bone marrow mesenchymal stem cells; granulocyte colony-stimulating factor; homing.