中国神经再生研究(英文版)

中国神经再生研究(英文版) ›› 2020, Vol. 15 ›› Issue (4): 690-696.doi: 10.4103/1673-5374.266920

• 原著:脊髓损伤修复保护与再生 • 上一篇    下一篇

表达HIF-1腺病毒感染骨髓间充质干细胞联合红景天苷保护急性脊髓损伤的作用

  

  • 出版日期:2020-04-15 发布日期:2020-05-28
  • 基金资助:

    国家高技术研究发展计划（863计划）（2015CB755400）

Protective effect of rhodioloside and bone marrow mesenchymal stem cells infected with HIF-1- expressing adenovirus on acute spinal cord injury

Xiao-Qin Ha1, Bo Yang2, 3, Huai-Jing Hou3, Xiao-Ling Cai4, Wan-Yuan Xiong3, Xu-Pan Wei3   

  1. 1 Lanzhou University Second Hospital, Lanzhou, Gansu Province, China
    2 Department of Clinical Laboratory, Lanzhou General Hospital of Lanzhou Military Area Command, Lanzhou, Gansu Province, China
    3 School of Clinical Medicine, Gansu University of Traditional Chinese Medicine, Lanzhou, Gansu Province, China
    4 School of Life Science and Engineering, Lanzhou University of Technology, Lanzhou, Gansu Province, China
  • Online:2020-04-15 Published:2020-05-28
  • Contact: Xiao-Qin Ha, MD,haxiaoqin2013@163.com; Bo Yang, Master candidate,1349151062@qq.com; Huai-Jing Hou, Doctoral candidate,757097029@qq.com.
  • Supported by:
    This study was supported by the National High Technology Research and Development Program of China (863 Program), No. 2015CB755400 (to XQH).

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摘要：

研究显示，红景天苷可保护细胞的缺氧损伤，骨髓间充质干细胞也有较好的修复效果。为了研究红景天苷联合骨髓间充质干细胞对脊髓损伤的作用，实验采用改良的Allen法建立大鼠脊髓损伤模型，建模后随机分为5组， PBS组采用PBS 5 μL灌胃，脊髓损伤部位5 mm周围、等距离6点、深度5 mm注射PBS；红景天苷组采用红景天苷灌胃(5 g/kg)，肌肉注射PBS；骨髓间充质干细胞组采用PBS灌胃，肌肉注射间充质干细胞(8×106/mL细胞悬液50 μL)；感染组采用PBS灌胃，肌肉注射含HIF-1腺病毒感染的骨髓间充质干细胞；联合组采用红景天苷灌胃，肌肉注射含HIF-1腺病毒感染的骨髓间充质干细胞。治疗后1周，采用改良的综合行为评分量表评价大鼠运动恢复情况。以苏木精伊红和Pischinger ' s亚甲蓝染色观察脊髓组织组织学和病理变化，采用实时荧光定量PCR技术检测脊髓组织腺病毒IX、间充质干细胞存活标记物Sry基因mRNA水平以确定转染进入脊髓腺病毒及间充质干细胞的数量，以免疫组织化学染色检测脊髓组织HIF-1蛋白水平。结果发现：(1)与其他3组相比较，红景天苷及联合组大鼠综合行为评分量表评分增高、组织损伤修复效果较好，Pischinger ' s亚甲蓝染色显示神经元数量较多；(2)与PBS组相比较，红景天组及联合组有更高的HIF-1免疫反应；(3)与HIF-1腺病毒感染的骨髓间充质干细胞组相比较，联合组脊髓组织间充质干细胞Sry基因mRNA水平增高；(4)结果证实，红景天苷联合骨髓间充质干细胞促进损伤脊髓的功能恢复，激活HIF-1通路促进骨髓间充质干细胞的存活和损伤脊髓组织神经元的修复。实验经甘肃中医学院动物伦理委员会2015年6月批准(批准号：2015KYLL029)。

orcid: 0000-0002-3440-9985 (Xiao-Qin Ha) 

         0000-0002-0772-7435 (Bo Yang) 

         0000-0003-2940-9670 (Huai-Jing Hou)

关键词: 急性脊髓损伤, 红景天苷, 骨髓间充质干细胞, 综合行为评分量表, HIF-1, IX基因, Sry基因, 神经修复, 腺病毒, 神经再生

Abstract: Rhodioloside has been shown to protect cells from hypoxia injury, and bone marrow mesenchymal stem cells have a good effect on tissue repair. To study the effects of rhodioloside and bone marrow mesenchymal stem cells on spinal cord injury, a rat model of spinal cord injury was established using the Infinite Horizons method. After establishing the model, the rats were randomly divided into five groups. Rats in the control group were intragastrically injected with phosphate buffered saline (PBS) (5 μL). PBS was injected at 6 equidistant points around 5 mm from the injury site and at a depth of 5 mm. Rats in the rhodioloside group were intragastrically injected with rhodioloside (5 g/kg) and intramuscularly injected with PBS. Rats in the mesenchymal stem cell (MSC) group were intramuscularly injected with PBS and intramuscularly with MSCs (8 × 106/mL in a 50-μL cell suspension). Rats in the Ad-HIF-MSC group were intragastrically injected with PBS and intramuscularly injected with HIF-1 adenovirus-infected MSCs. Rats in the rhodioloside + Ad-HIF-MSC group were intramuscularly injected with MSCs infected with the HIF-1 adenovirus and intragastrically injected with rhodioloside. One week after treatment, exercise recovery was evaluated with a modified combined behavioral score scale. Hematoxylin-eosin staining and Pischingert’s methylene blue staining were used to detect any histological or pathological changes in spinal cord tissue. Levels of adenovirus IX and Sry mRNA were detected by real-time quantitative polymerase chain reaction and used to determine the number of adenovirus and mesenchymal stem cells that were transfected into the spinal cord. Immunohistochemical staining was applied to detect HIF-1 protein levels in the spinal cord. The results showed that: (1) compared with the other groups, the rhodioloside + Ad-HIF-MSC group exhibited the highest combined behavioral score (P < 0.05), the most recovered tissue, and the greatest number of neurons, as indicated by Pischingert’s methylene blue staining. (2) Compared with the PBS group, HIF-1 protein expression was greater in the rhodioloside group (P < 0.05). (3) Compared with the Ad-HIF-MSC group, Sry mRNA levels were higher in the rhodioloside + Ad-HIF-MSC group (P < 0.05). These results confirm that rhodioloside combined with bone marrow mesenchymal stem cells can promote the recovery of spinal cord injury and activate the HIF-1 pathway to promote the survival of bone marrow mesenchymal stem cells and repair damaged neurons within spinal cord tissue. This experiment was approved by the Animal Ethics Committee of Gansu University of Traditional Chinese Medicine, China (approval No. 2015KYLL029) in June 2015.

Key words: acute spinal cord injury, adenovirus, adenovirus gene IX, bone marrow mesenchymal stem cells, combined behavioral score scale, HIF-1α, nerve regeneration, nerve repair, Rhodiola rosea, Sry