中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (27): 5028-5032.doi: 10.3969/j.issn.1673-8225.2010.27.020

• 干细胞因子及调控因子 stem cell factors and regulatory factors • 上一篇    下一篇

胶质细胞源性神经营养因子基因修饰CD34+细胞静脉移植治疗高血压大鼠脑梗死

欧雅莉，余国龙，杨天伦，方  立，胡  柯   

  1. 中南大学湘雅医院心内科，湖南省长沙市  410008
  • 出版日期:2010-07-02 发布日期:2010-07-02
  • 通讯作者: 余国龙，教授，中南大学湘雅医院心内科，湖南省长沙市 410008 yuguolong123@yahoo.com.cn
  • 作者简介:欧雅莉☆，女，1982年生，湖南省资兴市人，回族，2009年中南大学湘雅医院毕业，博士，医师，主要从事干细胞治疗缺血性疾病的研究。 ou-yali@hotmail.com
  • 基金资助:

    国家自然科学基金项目(30572079)。

Intravenous transplantation of glial cell-derived neurotrophic factor gene modified CD34+ cells for treating cerebral infarction in spontaneous hypertensive rats

Ou Ya-li, Yu Guo-long, Yang Tian-lun, Fang Li, Hu Ke   

  1. Department of Cardiology, Xiangya Hospital, Central South University, Changsha  410008, Hunan Province, China
  • Online:2010-07-02 Published:2010-07-02
  • Contact: Yu Guo-long, Professor, Department of Cardiology, Xiangya Hospital, Central South University, Changsha 410008, Hunan Province, China yuguolong123@ yahoo.com.cn
  • About author:Ou Ya-li☆, Doctor, Physician, Department of Cardiology, Xiangya Hospital, Central South University, Changsha 410008, Hunan Province, China ou-yali@hotmail.com
  • Supported by:

    the National Natural Science Foundation of China, No. 30572079*

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摘要：

背景：近年来，已有实验证实经颅直接给予胶质细胞源性神经营养因子(glial cell-derived neurotrophic factor，GDNF)或携带GDNF基因病毒载体有显著减少脑梗死体积、促进神经功能恢复的疗效，但其治疗方法有创，临床应用有限。
目的：观察GDNF基因转染的人脐血CD34+细胞经静脉移植对自发性高血压大鼠脑梗死的疗效，并探讨其机制。
方法：分离人脐血CD34+细胞，脂质体方法转染pEGFP-GDNF质粒和pEGFP空载体至CD34+细胞制备pEGFP-GDNF-CD34+和pEGFP-CD34+细胞；制备60只雄性自发性高血压大鼠大脑中动脉栓死模型并随机分为3组：pEGFP-GDNF-CD34+细胞移植组(基因细胞组)、pEGFP-CD34+细胞移植组(单纯细胞组)、生理盐水组。改良神经功能损害评分评价神经功能恢复状况；图像分析法观察TTC染色脑梗死体积；酶联免疫法检测细胞培养液与脑组织匀浆GDNF水平，荧光显微镜及免疫组织化学检测绿色荧光蛋白标记CD34+细胞及其人神经胶质纤维酸性蛋白和人神经元核抗原表达。
结果与结论：经静脉移植pEGFP-GDNF-CD34+细胞向自发性高血压大鼠脑缺血区域迁移、存活、并向神经细胞定向分化，促进神经功能恢复。GDNF基因转染CD34+细胞在脑组织存活、向神经细胞分化及对大脑神经功能保护作用优于CD34+细胞。脑组织GDNF水平可能是GDNF基因转染CD34+细胞和单纯CD34+细胞移植治疗自发性高血压大鼠局灶性脑缺血疗效差异机制之一。

关键词: 胶质细胞源性神经营养因子, CD34+细胞, 神经功能, 移植, 高血压大鼠

Abstract:

BACKGROUND: Previous studies have confirmed that direct administration of glial cell-derived neurotrophic factor (GDNF) or viral vector carrying GDNF gene can significantly reduce cerebral infarction volume, promote outcomes of the recovery of nerve function, but the therapeutic method is invasive and limits in clinic.
OBJECTIVE: To observe the outcomes of intravenous transplantation of human umbilical cord blood (hUCB) CD34+ cells transfected with GDNF gene in spontaneous hypertensive rats with cerebral infarction and to explore its mechanism.
METHODS: CD34+ cells isolated from hUCB were transfected by the pEGFP-GDNF plasmid and pEGFP blank vector to prepare pEGFP-GDNF-CD34+ and pEGFP-CD34+ cells. Sixty adult male spontaneous hypertensive rats with middle cerebral artery occlusion (MCAO) were randomly divided into three groups: pEGFP-GDNF-CD34+ cell group, pEGFP-CD34+ cell group and saline group. Neurological functional measurements were performed using the modified neurological severity score. Quantitative histological determinations of infarct volume were performed using standard TTC staining and quantitative image analysis. The GDNF level in the cell culture or the cerebral tissue was measured by enzyme linked immunosorbent assay. The survival and migration of green fluorescent protein (GFP)-labeled CD34+ cells and the expression of astrocytic marker-glial fibriliary acidic protein (GFAP) and the neuron marker-neuronal nuclei (NeuN) were detected by immunohistochemical and fluorescent staining.
RESULTS AND CONCLUSION: Intravenous transplantation of pEGFP-GDNF-CD34+ cells migrated into ischemic-injured cerebral hemisphere, survived and differentiated into neural cells in ischemic region of spontaneous hypertensive rats, and promoted the recovery of nerve function. GDNF gene-transfected CD34+ cells survived in the brain tissue and differentiated into neural cells, and its neurological protection effect was superior to CD34+ cells. The increased GDNF level in cerebral tissue was one of possible mechanisms responsible for focal cerebral ischemia in spontaneous hypertensive rats after transplantation of between GDNF gene modified CD34+ cells and CD34+ cells.

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