Therapeutic effect of bone marrow mesenchymal stem cell transplantation for spinal cord injury

doi:10.3969/j.issn.2095-4344.0411

Abstract

Abstract:

BACKGROUND: At present, treatments for spinal cord injury are limited, with poor outcomes. Therefore, it is of great importance to explore new treatment methods.
OBJECTIVE: To observe the therapeutic effect of bone marrow mesenchymal stem cells (BMSCs) injection via the caudal vein on spinal cord injury in rats.
METHODS: BMSCs were isolated and cultured in vitro by whole bone marrow adherence method. The surface markers were identified by flow cytometry. Thirty Sprague-Dawley rats were randomly divided into control group, spinal cord injury group and BMSCs transplantation group, 10 rats in each group. A rat spinal cord injury model was established by occlusion of the 10th thoracic vertebra using an aneurysm clamp, and 2×10⁶ BMSCs were injected through the caudal vein at 10 minutes after modeling. Basso-Bettle-Bresnahan (BBB) score for motor function recovery was assessed at 0, 10, 20, 30 days after transplantation. The therapeutic effect was evaluated by hematoxylin-eosin staining and electron transmission microscopy at 30 days after implantation.
RESULTS AND CONCLUSION: Under the microscope, fusiformis-shaped or fibroblast-like cells were observed. The expression rate of CD44 and CD90 was more than 90% and the expression of CD45 was less than 2%, by which, the BMSCs were identified. The BBB scores were significantly higher in the BMSCs transplantation group than the spinal cord injury group at 20 and 30 days after transplantation (P < 0.05). Hematoxylin-eosin staining showed that there was spinal cord tissue damage, vascular rupture injury, neuronal cell degeneration and inflammatory cell infiltration in the spinal cord injury group. After BMSCs transplantation, the number of spinal cord neurons was markedly increased with intact cytomembrane and clear nucleolus. Electron microscopic results showed that spinal cord axon swelling, demyelination, nerve axon deformation and necrosis were observed in the spinal cord injury group, while after BMSCs transplantation, the rat spinal cord axon structure was repaired, and partially lost myelin was recovered with uniform thickness. To conclude, BMSCs transplantation via the caudal vein has a significant therapeutic effect on spinal cord injury in rats by repairing the spinal cord structure and protecting the integrity of axon and myelin structures.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Bone Marrow, Mesenchymal Stem Cell Transplantation, Spinal Cord Injuries, Tissue Engineering

CLC Number:

R394.2

Sun Jing-song, Zhou Xue-ying, Qu Shu-xian, Li Shuang-yue. Therapeutic effect of bone marrow mesenchymal stem cell transplantation for spinal cord injury[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(1): 59-64.

Figures/Tables 1

2.1 骨髓间充质干细胞的形态骨髓细胞刚接种于培养皿时，细胞为大小不一的圆形，大多数悬浮在培养液中，6-8 h后悬浮的细胞开始贴壁，24 h后细胞基本完成贴壁(图1A)。换液去掉未贴壁的细胞，可见细胞分散分布并开始贴壁生长，呈梭形或多角形。四五天后可见细胞呈集落排列并伸出粗细、长短不一的突起，胞浆比较丰富，核仁清晰可见(图1B)。10 d左右可观察到细胞间隙变窄，出现相互融合现象，并按照胞体长轴有序的排列，整体呈螺旋形或漩涡状(图1C)。在进行稳定传代后，可观察到细胞形态和生长速度没有明显改变，多为不规则的梭形或者多角形，细胞核仁为圆形或者椭圆形(图1D)。 2.2 骨髓间充质干细胞表面标志物检测流式细胞仪检测结果显示，在培养的第3代大鼠骨髓间充质干细胞中，CD44和CD90的阳性表达率均大于90%，而CD45的表达率不到2%，见图2。 2.3 骨髓间充质干细胞移植对行为学评分的影响造模前，各组大鼠活动正常，BBB评分21分。造模后表现右后肢及尾无活动，针刺皮肤无反应，排尿障碍，排便未观察到明显异常，BBB评分为0分，移植后BBB评分见表1。 2.4 骨髓间充质干细胞移植对脊髓组织形态学的影响正常对照组大鼠脊髓灰质、白质结构清晰，界限明确，神经细胞形态正常，尼氏体可见，细胞膜和核仁结构完整，白质内可见神经纤维排列整齐，细胞间基质均一分布，未见水肿和炎细胞浸润，见图3A。脊髓损伤组大鼠脊髓灰白质界限不清，白质周围可见细胞肿胀及空泡样变性，细胞数目显著减少，细胞膜破坏严重，细胞核固缩、溶解和消失，并有大量的炎性细胞和巨噬细胞浸润，见图3B。与脊髓损伤组相比，骨髓间充质干细胞移植组大鼠脊髓组织形态显著改善，灰白质结构清晰，界限较清晰，神经细胞的数量明显增多，细胞膜结构完整，核仁清晰，见图3C。 2.5 骨髓间充质干细胞移植对脊髓组织超微结构的影响正常对照组大鼠脊髓轴索饱满且规则，轴突的周围均有髓鞘包绕，髓鞘的厚度均一，轴突的形态完整，见图4A。脊髓损伤组大鼠脊髓轴索变性、结构破坏，大部分髓鞘出现溶解和脱落，神经丝排列稀疏，轴突内出现空泡样改变，见图4B。骨髓间充质干细胞移植组大鼠脊髓中可观察到新生的轴突，并且结构完整，部分脱失的髓鞘得以修复和再生，见图4C。"

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