中国修复重建外科杂志

中国修复重建外科杂志

粒细胞集落刺激因子动员 BMSCs 归巢治疗大鼠脊髓损伤的疗效观察

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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 T10 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。

ObjectiveTo 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. MethodsTwenty-four healthy adult female Sprague Dawley rats were injected with 1 mL green fluorescence protein labeled BMSCs (GFP-BMSCs, 1×106 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 T10 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. ResultsThe 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). ConclusionG-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.

关键词: 脊髓损伤; 粒细胞集落刺激因子; BMSCs; 归巢

Key words: Spinal cord injury; granulocyte colony-stimulating factor; bone marrow mesenchymal stem cells; homing

引用本文: 李杰, 陈雷, 陈秋洪, 胡德庆, 林建华. 粒细胞集落刺激因子动员 BMSCs 归巢治疗大鼠脊髓损伤的疗效观察. 中国修复重建外科杂志, 2019, 33(1): 93-100. doi: 10.7507/1002-1892.201806127 复制

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1. Guo Y, Liu S, Wang P, et al. Granulocyte colony-stimulating factor improves neuron survival in experimental spinal cord injury by regulating nucleophosmin-1 expression. J Neurosci Res, 2014, 92(6): 751-760.
2. Chen WF, Chen CH, Chen NF, et al. Neuroprotective effects of direct intrathecal administration of granulocyte colony-stimulating factor in rats with spinal cord injury. CNS Neurosci Ther, 2015, 21(9): 698-707.
3. Kadota R, Koda M, Kawabe J, et al. Granulocyte colony-stimulating factor (G-CSF) protects oligodendrocyte and promotes hindlimb functional recovery after spinal cord injury in rats. PLoS One, 2012, 7(11): e50391.
4. Takahashi H, Yamazaki M, Okawa A, et al. Neuroprotective therapy using granulocyte colony-stimulating factor for acute spinal cord injury: a phase Ⅰ/Ⅱa clinical trial. Eur Spine J, 2012, 21(12): 2580-2587.
5. 李晓飞, 文益民, 张增山, 等. 骨髓基质干细胞移植联合应用 G-CSF 对大鼠脊髓损伤修复的影响. 中国矫形外科杂志, 2010, 18(17): 1457-1462.
6. Hibbert B, Hayley B, Beanlands RS, et al. Granulocyte colony-stimulating factor therapy for stem cell mobilization following anterior wall myocardial infarction: the CAPITAL STEM MI randomized trial. CMAJ, 2014, 186(11): E427-E434.
7. 梁少兰, 杜作义, 李自成, 等. 粒细胞集落刺激因子动员内皮祖细胞对心肌梗死患者心功能的影响. 实用医学杂志, 2014, 30(17): 2759-2761.
8. Shyu WC, Lin SZ, Lee CC, et al. Granulocyte colony-stimulating factor foracute ischemic stroke: arandomized controlled trial. CMAJ, 2006, 174(7): 927-933.
9. Nefussy B, Artamonov I, Deutsch V, et al. Recombinant human granulocyte-colony stimulating factor administration for treating amyotrophic lateral sclerosis: A pilot study. Amyotroph Lateral Scler, 2010, 11(1-2): 187-193.
10. Ito Y, Sugimoto Y, Tomioka M, et al. Does high dose methylprednisolone sodium succinate really improve neurological status in patient with acute cervical cord injury?: a prospective study about neurological recovery and early complications Spine (Phila Pa 1976), 2009, 34(20): 2121-2124.
11. Matsumoto T, Tamaki T, Kawakami M, et al. Early complications of high-dose methylprednisolone sodium succinate treatment in the follow-up of acute cervical spinal cord injury. Spine (Phila Pa 1976), 2001, 26(4): 426-430.
12. 杨瑞瑞, 邓宇斌. 干细胞修复脊髓损伤的研究现状. 实用医学杂志, 2017, 33(1): 10-13.
13. Woodbury D, Schwarz EJ, Prockop DJ, et al. Adult rat and humanbone marrow stromal cells differentiate into neurons. J Neurosci Res, 2000, 61(4): 364-370.
14. 韩博, 蒋玉东, 董大明, 等. 骨髓间充质干细胞治疗脊髓损伤的研究进展. 中华实用诊断与治疗杂志, 2017, 31(3): 300-302.
15. Tetzlaff W, Okon EB, Karimi-Abdolrezaee S, et al. A systematic review of cellular transplantation therapies for spinal cord injury. J Neurotrauma, 2011, 28(8): 1611-1682.
16. Platzbecker U, Prange-Krex G, Bornhäuser M, et al. Spleen enlargement in healthy donors during G-CSF mobilization of PBPCs. Transfusion, 2001, 41(2): 184-189.
17. Nishio Y, Koda M, Kamada T, et al. Granulocyte colony-stimulating factor attenuates neuronal death and promotes functional recovery after spinal cord injury in mice. J Neuropathol Exp Neurol, 2007, 66(8): 724-731.
18. Ha Y, Park HS, Park CW, et al. Synthes Award for Resident Research on Spinal Cord and Spinal Column Injury: granulocyte macrophage colony stimulating factor (GM-CSF) prevents apoptosis and improves functional outcome in experimental spinal cord contusion injury. Clin Neurosurg, 2005, 52: 341-347.
19. Guo X, Bu X, Li Z, et al. Comparison of autologous bone marrow mononuclear cells transplantation and mobilization by granulocyte colony-stimulating factor in experimental spinal injury. Int J Neurosci, 2012, 122(12): 723-733.
20. 张慧, 郭雨霁, 邴鲁军, 等. 粒细胞集落刺激因子对小鼠急性脊髓损伤的神经保护作用及其机制. 解剖学报, 2012, 43(6): 734-738.
21. Khorasanizadeh M, Eskian M, Vaccaro AR, et al. Granulocyte Colony-Stimulating Factor (G-CSF) for the Treatment of Spinal Cord Injury. CNS Drugs, 2017, 31(11): 911-937.
22. Deak E, Seifried E, Henschler R. Homing pathways of mesenchymal stromal cells (MSCs) and their role in clinical applications. Int Rev Immunol, 2010, 29(5): 514-529.
23. 郭卫春, 李军, 熊敏, 等. 基质细胞衍生因子-1/趋化因子受体 4 轴在促红细胞生成素动员骨髓间充质干细胞治疗脊髓损伤中的作用. 中华实验外科杂志, 2015, 32(7): 1506-1509.
24. Lin L, Lin H, Bai S, et al. Bone marrow mesenchymal stem cells (BMSCs) improved functional recovery of spinal cord injury partly by promoting axonal regeneration. Neurochem Int, 2018, 115: 80-84.
25. Kadota R, Koda M, Kawabe J, et al. Granulocyte colony-stimulating factor (G-CSF) protects oligodendrocyte and promotes hindlimb functional recovery after spinal cord injury in rats. PLoS One, 2012, 7(11): e50391.
26. Lee JS, Yang CC, Kuo YM, et al. Delayed granulocyte colony-stimulating factor treatment promotes functional recovery in rats with severe contusive spinal cord injury. Spine (Phila Pa 1976), 2012, 37(1): 10-17.
27. Chung J, Kim MH, Yoon YJ, et al. Effects of granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor on glial scar formation after spinal cord injury in rats. J Neurosurg Spine, 2014, 21(6): 966-973.
28. Okuda A, Horii-Hayashi N, Sasagawa T, et al. Bone marrow stromal cell sheets may promote axonal regeneration and functional recovery with suppression of glial scar formation after spinal cord transection injury in rats. J Neurosurg Spine, 2017, 26(3): 388-395.
29. Urdziková L, Likavčanová-Mašinová K, Vanĕček V, et al. Flt3 ligand synergizes with granulocyte-colony-stimulating factor in bone marrow mobilization toimprove functional outcome after spinal cord injury in the rat. Cytotherapy, 2011, 13(9): 1090-1104.
30. Kawabata H, Setoguchi T, Yone K, et al. High mobility group box 1 is upregulated after spinal cord injury and is associated with neuronal cell apoptosis. Spine (Phila Pa 1976), 2010, 35(11): 1109-1115.
31. Wu H, Lu D, Jiang H, et al. Simvastatin-mediated upregulation of VEGF and BDNF, activation of the PI3K/Akt pathway, and increase of neurogenesis are associated with therapeutic improvement after traumatic brain injury. J Neurotrauma, 2008, 25(21): 130-139.
32. Khorasanizadeh M, Eskian M, Vaccaro AR, et al. Granulocyte colony-stimulating factor (G-CSF) for the treatment of spinal cord injury. CNS Drugs, 2017, 31(11): 911-937.