中国组织工程研究

中国组织工程研究 ›› 2019, Vol. 23 ›› Issue (21): 3386-3391.doi: 10.3969/j.issn.2095-4344.1762

• 干细胞移植 stem cell transplantation • 上一篇    下一篇

经鼻腔移植人脐带间充质干细胞修复新生大鼠缺氧缺血性脑损伤

杨汉华1，田树凤2，谢丽春3，陈运彬4，马 廉5   

  1. 1深圳市坪山区妇幼保健院，深圳大学妇幼保健院新生儿科，广东省深圳市 518122；深圳市儿童医院，2感染科，3血液肿瘤科，广东省深圳市 518038；4广东省妇幼保健院，广东省广州市 510010；5深圳市儿童医院儿研所，广东省深圳市 518038)
  • 修回日期:2019-03-25 出版日期:2019-07-28 发布日期:2019-07-28
  • 通讯作者: 马廉，教授，博士生导师，主任医师，深圳市儿童医院儿研所，广东省深圳市 518038； 陈运彬，教授，博士生导师，主任医师，广东省妇幼保健院，广东省广州市 510010
  • 基金资助:

    深圳市知识创新计划项目(JCYJ20160429141742207)，项目负责人：杨汉华；国家自然科学基金(81671525)，项目负责人：马廉

Repairing hypoxic-ischemic brain damage in neonatal rats by nasal transplantation of human umbilical cord mesenchymal stem cells

Yang Hanhua1, Tian Shufeng2, Xie Lichun3, Chen Yunbin4, Ma Lian5   

  • Revised:2019-03-25 Online:2019-07-28 Published:2019-07-28
  • Contact: Ma Lian, Professor, Doctoral supervisor, Chief physician, Institute of Pediatrics, Shenzhen Children’s Hospital, Shenzhen 518038, Guangdong Province, China; Chen Yunbin, Professor, Doctoral supervisor, Chief physician, Guangdong Maternal and Child Health Hospital, Guangzhou 510010, Guangdong Province, China
  • Supported by:

    Shenzhen Knowledge Innovation Program, No. JCYJ20160429141742207 (to YHH); the National Natural Science Foundation of China, No. 81671525 (to ML)

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摘要：

文章快速阅读：

文题释义：
经鼻腔移植人脐带间充质干细胞：通过鼻腔越过血脑屏障，将1×106人脐带间充质干细胞直接给到中枢神经系统。经鼻腔移植细胞的优势在于：细胞可通过嗅神经、三叉神经等途径直接进入到脑组织，所需细胞量小，无创伤性，同时可减少系统给药的不良反应。
人脐带间充质干细胞修复新生儿缺血性脑损伤：人脐带间充质干细胞可促进实验动物缺血性脑损伤后神经功能恢复，可减少缺氧缺血性脑损伤新生大鼠神经细胞凋亡并改善其记忆学习能力，而且，人脐带间充质干细胞具有来源丰富、容易提取、倍增时间短、免疫原性低、移植后长时间存活、不涉及伦理问题等多种优点，作为治疗新生儿缺氧缺血性脑损伤的细胞来源有着广泛的应用前景。

 

摘要
背景：动物实验显示人脐带间充质干细胞移植具有显著的神经保护作用，其移植途径包括经静脉、动脉、腰穿、脑立体定向移植和侧脑室移植多种，均存在不足。
目的：探讨通过鼻腔移植人脐带间充质干细胞修复新生大鼠缺氧缺血性脑损伤的可行性和有效性。
方法：取90只7 d龄SD大鼠(汕头大学医学院实验动物中心提供)，随机分3组，每组30只：假手术组分离左颈总动脉后缝合皮肤；模型组采用结扎切断左颈总动脉联合缺氧仓的方法中制备缺氧缺血性脑损伤模型；实验组采用同样方法制备缺氧缺血性脑损伤模型，同时鼻腔滴入绿色荧光蛋白标记的人脐带间充质干细胞。建模后3 d取脑组织，进行尼氏染色、TUNEL染色及免疫荧光染色；建模后14，29 d，水迷宫实验检测大鼠记忆、学习能力，14 d水迷宫实验完成后行脑组织TUNEL染色。动物实验经深圳大学医学部实验动物伦理委员会批准(伦理批准号：2016-121)。
结果与结论：①尼氏染色：模型组大脑皮质及海马CA1区神经元排列均不规则，尼氏小体数量少；实验组大鼠大脑皮质及海马CA1区神经元排列较模型组规则，尼氏小体数量较多；②TUNEL染色：建模后3，14 d，模型组凋亡细胞数多于假手术组(P < 0.05)，实验组凋亡细胞数少于模型组(P < 0.05)；③免疫荧光染色：实验组脑组织中可见绿色标记的人脐带间充质干细胞，主要分布于损伤侧海马及大脑皮质区域，非损伤侧脑组织偶可见绿色标记细胞，未见绿色荧光与GFAP或NSE染色双阳性细胞；④实验组建模后14，29 d的平均逃逸潜伏期均低于模型组(P < 0.05)；⑤结果表明：经鼻腔移植人脐带间充质干细胞修复新生大鼠缺氧缺血性脑损伤，可减少神经细胞凋亡，改善大鼠记忆学习能力。


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: 0000-0001-6378-0217(杨汉华)

关键词: 干细胞, 脐带干细胞, 间充质干细胞, 脐带间充质干细胞, 移植, 缺氧缺血性脑损伤, 鼻腔给药, 水迷宫实验

Abstract:

BACKGROUND: Human umbilical cord mesenchymal stem cells exert significant neuroprotective effects in animals, including reducing neuronal apoptosis and promoting neuronal regeneration. Current approaches to transfer these cells include transvenous, arterial, lumbar puncture, brain stereotactic transplantation and lateral ventricle transplantation, but all of them are insufficient. 
OBJECTIVE: To investigate the feasibility and effectiveness of nasal transplantation of human umbilical cord mesenchymal stem cells in the treatment of hypoxic-ischemic brain damage in neonatal rats.
METHODS: Ninety 7-day-old Sprague-Dawley rats (provided by the Experimental Animal Center of Shantou University Medical College) were randomly divided into three groups (n=30 per group): in sham operation group the left common carotid artery was only separated and then sutured; in model group, an animal model of hypoxic-ischemic brain damage was prepared through ligation and occlusion of the left common carotid artery combined with hypoxic chamber; in experimental group, the animal model was prepared as described in the model group, and then green fluorescent protein-labeled human umbilical cord mesenchymal stem cells were instilled into the nasal cavity of model rats. Brain tissue was taken 3 days after modeling, and Nissl staining, TUNEL detection, and immunofluorescence staining were performed. The memory and learning abilities of the rats were detected by water maze at 14 and 29 days after modeling. After 14-day water maze test, TUNEL staining of the brain tissue was performed. The animal experiment was approved by the Experimental Animal Ethics Committee of Shenzhen University Health Science Center with the approval No. 2016-121.
RESULTS AND CONCLUSION: (1) Nissl staining: Neurons in the cerebral cortex and hippocampal CA1 area of the model group were irregularly arranged, and there were few Nissl bodies. Compared with the model group, the neurons in the cerebral cortex and hippocampal CA1 area were arranged more regularly in the experimental group, and there were more Nissl bodies. (2) TUNEL staining: On 3 and 14 days after modeling, the number of apoptotic cells in the model group was significantly higher than that in the sham operation group (P < 0.05) and in the experimental group (P < 0.05). (3) Immunofluorescence staining: Green-labeled human umbilical cord mesenchymal stem cells were found in the brain tissue of the experimental group, mainly distributed in the hippocampus and cerebral cortex of the injured side. Green fluorescent protein-labeled cells were mainly visible in the injured hippocampus and cortex, but rarely detected in the non-injured brain tissues. Non-GFAP or NSE double staining cells were detected. (4) The average escape latency of the experimental group was lower than that of the model group (P < 0.05). Overall, these findings indicate that nasal transplantation of human umbilical cord mesenchymal stem cells can reduce neuronal apoptosis and improve memory and learning abilities of neonatal rats with hypoxic-ischemic brain damage.

Key words: stem cells, umbilical cord blood stem cells, mesenchymal stem cells, umbilical cord blood mesenchymal stem cells, transplantation, hypoxic-ischemic brain damage, intranasal delivery, water maze test

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