中国组织工程研究

中国组织工程研究

• 干细胞培养与分化 • 上一篇    下一篇

小胶质细胞活化刺激骨髓间充质干细胞释放胶质细胞源性神经营养因子保护多巴胺能神经元的实验

范东艳1，王 苹2，刘 然1，3，牛凤兰1，杜 波2   

  1. 1吉林大学公共卫生学院毒理学教研室，吉林省长春市  130021；
    2吉林大学第一临床医院耳鼻喉-头颈外科，吉林省长春市  130021；
    3长春市中心血站，吉林省长春市  130000
  • 出版日期:2010-02-05 发布日期:2010-02-05
  • 通讯作者: 杜 波，博士，副教授，吉林大学第一临床医院耳鼻喉-头颈外科，吉林省长春市 130021 dubo2222@hotmail.com
  • 作者简介:范东艳，女，1973年生，吉林省吉林市人，汉族，吉林大学在读博士，主要从事干细胞性质及应用的研究。 Dongyan_fan@yahoo.com.cn

Microglia activation stimulates bone marrow mesenchymal stem cells to release gliocyte-derived neurotrophic factor for protection of dopaminergic neurons

Fan Dong-yan1, Wang Ping2, Liu Ran1, 3, Niu Feng-lan1, Du Bo2   

  1. 1Teaching and Research Section of Toxicology, College of Public Health, Jilin University, Changchun   130021, Jilin Province, China;
    2Department of Otolaryngology, Head and Neck Surgery, First Clinical Hospital, Jilin University, Changchun   130021, Jilin Province, China;
    3Changchun Blood Center, Changchun   130000, Jilin Province, China
  • Online:2010-02-05 Published:2010-02-05
  • Contact: Du Bo, Doctor, Associate professor, Department of Otolaryngology, Head and Neck Surgery, First Clinical Hospital, Jilin University, Changchun 130021, Jilin Province, China dubo2222@hotmail.com
  • About author:Fan Dong-yan, Studying for doctorate, Teaching and Research Section of Toxicology, College of Public Health, Jilin University, Changchun 130021, Jilin Province, China Dongyan_fan@yahoo.com.cn

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559

被引次数

13

摘要：

背景：目前尚未见骨髓间充质干细胞对活化的小胶质细胞特异性反应的报道，且有关骨髓间充质干细胞在特定微环境下如何维持多巴胺能神经元的存活也缺乏相应的实验证据。

目的：观察骨髓间充质干细胞在活化的小胶质细胞刺激下保护多巴胺能神经元存活的作用。

方法：取Wistar大鼠，贴壁法分离培养骨髓间充质干细胞，体外培养并活化小胶质细胞，酶消化法培养中脑多巴胺能神经元。实验分为5组：骨髓间充质干细胞组；小胶质细胞组；脂多糖+小胶质细胞组；骨髓间充质干细胞+脂多糖+小胶质细胞组；分别取各实验组的培养上清，对中脑多巴胺神经元进行培养。单纯多巴胺能神经元组采用体积分数为10%胎牛血清+DMEM/F12进行培养。采用免疫荧光技术检测不同微环境对多巴胺能神经元存活的影响及不同微环境对骨髓间充质干细胞释放胶质细胞源性神经营养因子的影响。

结果与结论：含有骨髓间充质干细胞的实验组胶质细胞源性神经营养因子的释放量均较相应的对照组高。酪氨酸羟化酶免疫荧光染色结果发现，单纯多巴胺能神经元组神经元的存活率为15%；小胶质细胞组多巴胺能神经元的存活率为10%；骨髓间充质干细胞组多巴胺能神经元的存活率为35%；脂多糖+小胶质细胞组多巴胺能神经元的存活率为5%；而骨髓间充质干细胞+脂多糖+小胶质细胞组多巴胺能神经元的存活率达到了28%，高于除骨髓间充质干细胞组外的其他各组(P < 0.05)。此外体外培养多巴胺能神经元存活率随培养时间延长下降，但含有骨髓间充质干细胞实验组的多巴胺能神经元存活率明显高于相应对照组。提示小胶质细胞活化刺激骨髓间充质干细胞上调胶质细胞源性神经营养因子表达，使得多巴胺能神经元免受毒素的损害，抑制了多巴胺能神经元的延迟性死亡。

关键词: 多巴胺能神经元, 骨髓间充质干细胞, 胶质细胞源性神经营养因子, 小胶质细胞, 帕金森病

Abstract:

BACKGROUND: Studies are very few regarding the specific reaction of bone marrow mesenchymal stem cells (BMSCs) to activated microglia. Moreover, it remains unclear how MSCs maintain dopaminergic neuronal survival under specific microenvironment.

OBJECTIVE: To explore the effect of BMSCs stimulated by activated microglia on dopaminergic neuron survival.

METHODS: BMSCs were isolated from Wistar rats by attachment method, and in vitro cultured; microglia was activated, and dopaminergic neurons were cultured by enzyme digestion method. The experiment included 5 groups: BMSCs, microglia, lipopolysaccharide (LPS)+microglia; BMSCs+LPS+microglia groups, in which the dopaminergic neurons were cultured with corresponding culture medium; the dopaminergic neurons alone group was cultured with 10% fetal bovine serum+ DMEM/F12. The effect of different microenvironment on dopaminergic neuron survival and gliocyte-derived neurotrophic factor released from BMSCs were detected by immunofluorescence technique.

RESULTS AND CONCLUSION: The release of gliocyte-derived neurotrophic factor in groups involving BMSCs was greater than corresponding control group. Tyrosine hydroxylase immunofluorescence showed that neuronal survival of dopaminergic neurons alone group was 15%, microglia group was 10%, LPS+microglia was 5%, but BMSCs+LPS+microglia group was 28%, significantly greater than the other groups (P < 0.05). In addition, survival of in vitro cultured dopaminergic neurons was decreased with increasing culture duration, but the survival of dopaminergic neurons in group involving BMSCs was significantly greater than corresponding control group. This indicates that microglia activation stimulated BMSCs to upregulate gliocyte-derived neurotrophic factor to prevent dopaminergic neurons from toxic injury, and inhibit delayed death of dopaminergic neurons.

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