中国组织工程研究

中国组织工程研究 ›› 2022, Vol. 26 ›› Issue (1): 20-26.doi: 10.12307/2022.004

• 骨髓干细胞 bone marrow stem cells • 上一篇    下一篇

骨髓间充质干细胞来源外泌体与硫酸软骨素酶ABC联合治疗大鼠脊髓损伤

谢兴奇，胡  伟，屠冠军   

  1. 中国医科大学附属第一医院骨科，辽宁省沈阳市  110000
  • 收稿日期:2021-02-05 修回日期:2021-02-07 接受日期:2021-03-13 出版日期:2022-01-08 发布日期:2021-10-23
  • 通讯作者: 屠冠军，博士，教授，主任医师，中国医科大学附属第一医院骨科，辽宁省沈阳市 110000
  • 作者简介:谢兴奇，男，1993年生，广东省阳春市人，汉族，中国医科大学在读硕士，主要从事间充质干细胞治疗脊髓损伤的研究。
  • 基金资助:
    辽宁省自然科学基金指导计划项目(201602857)，项目负责人：屠冠军

Bone marrow mesenchymal stem cells-derived exosomes combined with chondroitinase ABC for treating spinal cord injury in rats

Xie Xingqi, Hu Wei, Tu Guanjun   

  1. Department of Orthopedics, First Affiliated Hospital of China Medical University, Shenyang 110000, Liaoning Province, China
  • Received:2021-02-05 Revised:2021-02-07 Accepted:2021-03-13 Online:2022-01-08 Published:2021-10-23
  • Contact: Tu Guanjun, MD, Professor, Chief physician, Department of Orthopedics, First Affiliated Hospital of China Medical University, Shenyang 110000, Liaoning Province, China
  • About author:Xie Xingqi, Master candidate, Department of Orthopedics, First Affiliated Hospital of China Medical University, Shenyang 110000, Liaoning Province, China
  • Supported by:
    the Guidance Program of the Natural Science Foundation of Liaoning Province, No. 201602857 (to TGJ) 

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摘要：


文题释义：

外泌体：是一种胞外囊泡，因携带蛋白质、脂质、RNA等生物活性物质，可调节细胞微环境及靶细胞的生物学行为。
生长相关蛋白43：是神经组织特有的胞膜磷酸蛋白，与脊髓损伤后神经元和轴突再生密切相关。

背景：如何改善损伤局部微环境，减少胶质瘢痕形成，促进神经元和轴突修复与再生，是治疗脊髓损伤的难点。研究发现，硫酸软骨素酶ABC可通过介导硫酸软骨素蛋白聚糖的降解，增强轴突的再生和可塑性；同时骨髓间充质干细胞来源外泌体可通过抗凋亡、调节免疫反应、增强神经元再生能力等途径在脊髓损伤修复中发挥作用。
目的：研究骨髓间充质干细胞来源外泌体联合硫酸软骨素酶ABC对大鼠脊髓损伤的修复作用，并探讨其机制。
方法：用外泌体提取试剂盒提取骨髓间充质干细胞外泌体，透射电镜观察外泌体形态，Western blot检测CD63、Alix表达。取50只SD大鼠随机分成5组：假手术组、模型组、硫酸软骨素酶ABC组、外泌体组、联合组，每组10只。采用改良Allen’s法构建大鼠脊髓损伤模型，硫酸软骨素酶ABC组、联合组用微量注射器直接向损伤局部注射硫酸软骨素酶ABC，术后2 h，外泌体组、联合组通过尾静脉注射骨髓间充质干细胞来源外泌体，每隔2 d注射1次，共注射3次。脊髓损伤后第1，3，7，14，28天进行后肢运动功能评分，第14天每组取6只大鼠经灌注取材做成石蜡切片，苏木精-伊红染色观察脊髓损伤区病理变化，免疫荧光检测胶质纤维酸性蛋白表达，免疫组化检测生长相关蛋白43表达。

结果与结论：①外泌体呈圆形、椭圆形或“茶托样”，大小不均；CD63、Alix呈阳性表达；②与模型组、硫酸软骨素酶ABC组、外泌体组相比，联合组BBB评分高，脊髓损伤区面积小，胶质纤维酸性蛋白表达降低，生长相关蛋白43表达升高(P均 < 0.05)；③结果表明，骨髓间充质干细胞外泌体联合硫酸软骨素酶ABC能抑制损伤区胶质纤维酸性蛋白表达，上调生长相关蛋白43表达，减少胶质瘢痕形成，改善损伤局部微环境，从而促进大鼠脊髓损伤后神经元和轴突的修复与再生。

https://orcid.org/0000-0002-0469-0032(谢兴奇) 

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

关键词: 干细胞, 骨髓间充质干细胞, 外泌体, 硫酸软骨素酶ABC, 脊髓损伤, 修复与再生, 组织工程, 胶质纤维酸性蛋白, 生长相关蛋白43

Abstract: BACKGROUND: How to improve the local microenvironment, inhibit the formation of the glial scar, and promote repair and regeneration of damaged neurons and axons, are the difficulties of treating spinal cord injury. The research demonstrated that chondroitinase ABC can enhance the regeneration and plasticity of axons by degrading chondroitin sulfate proteoglycans. Meanwhile, bone marrow mesenchymal stem cells-derived exosome has a protective effect on spinal cord injury recovery by inhibiting apoptosis, regulating immune response, and regulating regeneration of damaged neurons.
OBJECTIVE: To investigate the repair effect of bone marrow mesenchymal stem cells-derived exosome combined with chondroitinase ABC on spinal cord injury in rats and to explore its mechanism.
METHODS:  Using the exosomes-extracted kit to isolate bone marrow mesenchymal stem cells-derived exosome, its morphology was observed under a transmission electron microscope. The expression levels of CD63 and Alix were identified through western blot assay. Totally 50 SD rats were randomly divided into five groups: sham group, model group, chondroitinase ABC group, exosome group, and combination group, with 10 rats in each group. Rat models of spinal cord injury were established by referencing the modified Allen’s method. In the chondroitinase ABC group and combination group, a micro syringe was applied to directly inject chondroitinase ABC into the injured area. At 2 hours after surgery, the models of the exosome group and the combination group were injected with bone marrow mesenchymal stem cells-derived exosomes through the tail vein, once every 2 days, for a total of 3 injections. The hind limb motor function was scored at 1, 3, 7, 14, and 28 days after surgery. At 14 days, six rats were taken from each group to make paraffin slices after perfusion. The pathological changes were observed at the injury region by hematoxylin-eosin staining. The expression of glial fibrillary acidic protein was detected by immunofluorescence and the expression of growth-associated protein-43 was identified by immunohistochemistry.   
RESULTS AND CONCLUSION: (1) The exosomes presented a circular or elliptical shape or “saucer-like” and its size was uneven. The positive expression of its marker proteins CD63 and Alix could be observed. (2) Compared with the model, chondroitinase ABC, and exosome groups, BBB score was higher; damaged area was smaller; expression of glial fibrillary acidic protein was lower; the expression of growth-associated protein-43 was up-regulated in the combination group (all P < 0.05). (3) The results demonstrate that bone marrow mesenchymal stem cells-derived exosome combined with chondroitinase ABC can inhibit the expression of glial fibrillary acidic protein in the injured area, up-regulate the expression of growth-associated protein-43, and reduce the formation of the glial scar, which improve local microenvironment and promote the repair and regeneration of neurons and axons after spinal cord injury in rats.

Key words: stem cells, bone marrow mesenchymal stem cells, exosomes, chondroitinase ABC, spinal cord injury, repair and regeneration, tissue engineering, glial fibrillary acidic protein, growth-associated protein-43

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