中国组织工程研究

中国组织工程研究 ›› 2019, Vol. 23 ›› Issue (21): 3294-3301.doi: 10.3969/j.issn.2095-4344.1760

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

骨髓间充质干细胞外泌体可减少脊髓损伤后A1型星形胶质细胞的活化

周 燕1，王 琳2，裴 双2，李燕飞2，陈雪梅3，贾延劼2   

  1. 郑州大学第一附属医院，1放射科，2神经内科，河南省郑州市 450052；3郑州大学基础医学院人体解剖学系，河南省郑州市 450001
  • 修回日期:2019-02-01 出版日期:2019-07-28 发布日期:2019-07-28
  • 通讯作者: 贾延劼，博士，博士生导师，教授，主任医师，郑州大学第一附属医院神经内科，河南省郑州市 450052
  • 作者简介:周燕，女，1974年生，陕西省西安市人，汉族，硕士，讲师，主要从事干细胞与医学影像学方面的研究。
  • 基金资助:

    国家自然科学基金(U1604170)，项目负责人：贾延劼

Bone marrow mesenchymal stem cell-derived exosomes reduce the activation of type A1 astrocytes after spinal cord injury

Zhou Yan1, Wang Lin2, Pei Shuang2, Li Yanfei2, Chen Xuemei3, Jia Yanjie2   

  1. 1Department of Radiology, 2Department of Neurology, the First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, Henan Province, China; 3Department of Human Anatomy, School of Basic Medical Sciences, Zhengzhou University, Zhengzhou 450001, Henan Province, China
  • Revised:2019-02-01 Online:2019-07-28 Published:2019-07-28
  • Contact: Jia Yanjie, MD, Doctoral supervisor, Professor, Chief physician, Department of Neurology, the First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, Henan Province, China
  • About author:Zhou Yan, Master, Lecturer, Department of Radiology, the First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, Henan Province, China
  • Supported by:

    the National Natural Science Foundation of China, No. U1604170 (to JYJ)

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摘要：

文章快速阅读：

文题释义：
外泌体：各种干细胞都可以分泌大量的外泌体，直径为40-100 nm，物理化学性质稳定，生物相容性好，是干细胞移植新的载体。
A1型胶质细胞：中枢神经系统损伤后，星形胶质细胞会从静息态向A1型转化，A1型胶质细胞引起细胞凋亡，抑制轴突生长等负向作用。

 

摘要
背景：课题组前期研究发现，骨髓间充质干细胞来源外泌体可减少脊髓损伤区域反应性星形胶质细胞的数量，有利于损伤后运动功能的改善。
目的：探索骨髓间充质干细胞外泌体对脊髓损伤后A1型星形胶质细胞活化的影响及其在损伤后修复中的作用。
方法：培养SD大鼠骨髓间充质干细胞，利用超速离心法提取细胞上清液中的外泌体并进行鉴定。将80只成年雄性SD大鼠随机分为假手术组、脊髓损伤模型组、骨髓间充质干细胞组、骨髓间充质干细胞外泌体组，每组20只。除假手术组外，其余3组均使用Allen法制备脊髓损伤模型，术后2 h通过尾静脉注射等体积的PBS、骨髓间充质干细胞悬液、骨髓间充质干细胞外泌体悬液，每隔2 d注射1次，共注射3次。脊髓损伤术前、术后1，3，7，14，28 d采用BBB评分法评估后肢运动功能；术后7 d通过苏木精-伊红染色、透射电镜观察脊髓组织形态，免疫荧光检测A1型星形胶质细胞数量、NeuN阳性神经元数量、caspase-3阳性凋亡细胞数量，ELISA法检测脊髓组织肿瘤坏死因子α、白细胞介素1α水平，Western blot检测脊髓组织C3、GFAP的表达水平。
结果与结论：①骨髓间充质干细胞外泌体在透射电镜下呈茶托状，表达外泌体标志CD63、CD9；②骨髓间充质干细胞外泌体减少脊髓组织炎症细胞浸润、减轻髓鞘脱失、轴突退变，降低炎症因子肿瘤坏死因子α、白细胞介素1α水平和C3的表达水平，降低A1型星形胶质细胞数量，减少细胞凋亡，提高残余神经元数量，改善肢体运动功能；③以上结果提示，骨髓间充质干细胞外泌体能减少脊髓A1型星形胶质细胞的活化，促进脊髓损伤后运动功能恢复。


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
ORCID:0000-0001-9571-6295(周燕)；0000-0002-2220-9257(贾延劼)

关键词: 脊髓损伤, 骨髓间充质干细胞, 外泌体, A1型星形胶质细胞, 炎症因子, 运动功能, 国家自然科学基金

Abstract:

BACKGROUND: Previous studies found that exosomes from bone marrow mesenchymal stem cells could reduce the number of reactive astrocytes after spinal cord injury, thereby improving post-injury motor function.
OBJECTIVE: To explore the role of bone marrow mesenchymal stem cell-derived exosomes in the activation of A1 astrocytes as well as in the repair of spinal cord injury. 
METHODS: Bone marrow mesenchymal stem cells from Sprague-Dawely rats were cultured, and then the exosomes from the cell supernatant were extracted and identified by ultracentrifugation. Eighty adult male Sprague-Dawley rats were randomly divided into sham operation group, spinal cord injury group (model group), bone marrow mesenchymal stem cell group (cell transplantation group) and exosome group (n=20/group). Except the sham operation group, an animal model of spinal cord injury was made in all the groups by Allen method. Two hours postoperatively, the equal volume of PBS, bone marrow mesenchymal stem cell suspension or bone marrow mesenchymal stem cell-derived exosome suspension was injected through the tail vein respectively, every 3 days, for three sessions. Basso, Beattie and Bresnahan scoring for motor function of the hind limb was performed preoperatively and and 1, 3, 7, 14, 28 days postoperatively. Hematoxylin-eosin staining and transmission electron microscope were used to observe the morphology of spinal cord tissue at 7 days postoperatively. Immunofluorescence staining was used to detect the number of A1 astrocytes, NeuN positive neurons and Caspase-3 positive apoptotic cells. ELISA assay was used to detect the levels of tumor necrosis factor-α and interleukin-1α in the spinal cord tissue. Western blot assay was used to determine relative protein expression levels of C3 and GFAP.
RESULTS AND CONCLUSION: Exosomes from bone marrow mesenchymal stem cells showed a saucer-like appearance under transmission electron microscope, expressing exosome markers CD63 and CD9. The exosomes reduced inflammatory cell infiltration of spinal cord tissue, reduced myelin degeneration and axonal degeneration, reduced the expression levels of tumor necrosis factor-α, interleukin-1α and C3, reduced the number of A1 astrocytes, and reduced cell apoptosis, but increased the number of residual neurons, and improved limb motor function. To conclude, exosomes from bone marrow mesenchymal stem cells can reduce the activation of type A1 astrocytes in the spinal cord and promote the recovery of motor function after spinal cord injury.

Key words: spinal cord injury, bone marrow mesenchymal stem cells, exosomes, type A1 astrocytes, inflammatory factors, motor function, National Natural Science Foundation of China

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