骨髓间充质干细胞细胞周期同步化的方法及对向神经细胞分化的影响

doi:10.3969/j.issn.1006-9771.2016.12.007

《中国康复理论与实践》 ›› 2016, Vol. 22 ›› Issue (12): 1399-1398.doi: 10.3969/j.issn.1006-9771.2016.12.007

骨髓间充质干细胞细胞周期同步化的方法及对向神经细胞分化的影响

李曼丽¹，赵文²，高钰丹²，段红梅¹，杨朝阳²，李晓光¹,²

1. 北京航空航天大学生物与医学工程学院，北京市 100191；
2.首都医科大学基础医学院，北京市 100069。

收稿日期:2016-06-17 出版日期:2016-12-25 发布日期:2017-01-15
通讯作者: 李晓光,E-mail: lxgchina@sina.com。
作者简介:李曼丽(1985-)，女，汉族，河北安新县人，博士研究生，主要研究方向：干细胞的诱导分化。
基金资助:
1.国家“863”计划项目(No.2012AA020206)；2.国家自然科学基金面上项目(No.31271037)；3.国家自然科学基金国际合作与交流项目(No.31320103903)；4.“十二五”国家科技支撑计划项目(No.2012BAI17B04)；5.高等学校全国优秀博士学位论文作者专项资金资助项目(No.201356)；6.国家国际科技合作专项项目(No.2014DFA30640)；7.国家自然科学基金委员会资助项目(No.31130022)。

Cell Cycle Synchronization Methods of Bone Marrow Mesenchymal Stem Cells and Its Effect on Neural Differentiation　

LI Man-li¹, ZHAO Wen², GAO Yu-dan², DUAN Hong-mei¹, YANG Zhao-yang², LI Xiao-guang¹,²

1. Department of Biomedical Engineering, School of Biological Science and Medical Engineering, Beihang University, Beijing 100191, China；
2. Department of Neurobiology, Capital Medical University, Beijing 100069, China

Received:2016-06-17 Published:2016-12-25 Online:2017-01-15
Contact: LI Xiao-guang. E-mail: lxgchina@sina.com

摘要/Abstract

摘要： 目的　分析不同处理条件下细胞同步化于G0/G1时期的效果以得到最佳的同步化条件，并研究同步化对骨髓间充质干细胞(BMSCs)在碱性成纤维细胞因子(bFGF)的作用下分化为神经细胞的影响。方法　分离培养成年大鼠BMSCs，5%、1%、0.5%、0.1%、0胎牛血清(FBS)分别处理24 h、48 h。PI染色后经流式细胞仪检测细胞周期各时相细胞所占比例，与正常培养条件(10% FBS)处理下对比。得到最佳处理条件后，bFGF处理3 d、7 d，免疫荧光细胞化学方法检测Nestin和Tuj-1的表达。结果　成年大鼠BMSCs原代提取，经传代后，细胞形态为长梭形。不同处理条件下G1/G0期细胞比例均高于正常培养条件，1% FBS处理48 h时G1/G0期细胞比例为(94.274±0.468)%，达最高峰(F=39.91, P<0.001)。bFGF诱导3 d后，细胞周期同步化后的Nestin+细胞数显著高于未同步化的细胞数[(80.3±2.4)% vs. (12.1±1.5)%] (F=28.25, P<0.001)；bFGF诱导7 d后，同步化后的Tuj-1+细胞数显著高于未同步化的细胞数[(74.8±3.2)% vs. (19.3±2.5)%] (F=17.95, P<0.001)。结论　1% FBS处理48 h是将BMSCs同步化于G0/G1期的最佳条件。细胞周期同步化于G0/G1期能够提高BMSCs向神经细胞分化的比例。

关键词: 骨髓间充质干细胞, 细胞周期, 同步化, 血清饥饿法, 神经分化, 大鼠

Abstract: Objective To analyze the effect of different treatment conditions on cells synchronization in G0/G1 phase to get the best condition, and to explore its effect on neural differentiation of bone marrow mesenchymal stem cells (BMSCs) induced by basic fibroblast growth factor (bFGF).Methods BMSCs were isolated and cultured in 5%, 1%, 0.5%, 0.1%, 0 fetal bovine serum (FBS) respectively, for 24 hours and 48 hours. After PI staining, cell cycle proportions of each phase were detected by flow cytometry, and were compared with the normal group (10% FBS). After the optimal treatment condition was got, 20 ng/ml bFGF was added into synchronization group and unsynchronization group 3 days and 7 days, respectively. The expression of Nestin and Tuj-1 were detected with immunofluorescence.Results Adult rat BMSCs were isolated from bone marrow and cultured, after passage, the cells were with long spindle shape. Compared with the normal group, the cell proportion of G1/G0 phase increased under different treatments, peaked with (94.274±0.468)% under 1% FBS, 48 hours (F=39.91, P<0.001). After bFGF induction for 3 days, the Nestin+ cell number was higher in the synchronization group than in the unsynchronization group [(80.3±2.4)% vs. (12.1±1.5)%] (F=28.25, P<0.001). After bFGF induction for 7 days, the Tuj-1+ cell number was higher in the synchronization group than in the unsynchronization group [(74.8±3.2%)% vs. (19.3±2.5)%] (F=17.95, P<0.001).Conclusion 1% FBS, 48 hours is the optimal condition to BMSCs synchronization in G0/G1 phase, which can promote the neural differentiation of BMSCs.

Key words: bone marrow mesenchymal stem cells, cell cycle, synchronization, serum starvation, neural differentiation, rats

中图分类号:

R329.2

李曼丽，赵文，高钰丹，段红梅，杨朝阳，李晓光,. 骨髓间充质干细胞细胞周期同步化的方法及对向神经细胞分化的影响[J]. 《中国康复理论与实践》, 2016, 22(12): 1399-1398.

LI Man-li, ZHAO Wen, GAO Yu-dan, DUAN Hong-mei, YANG Zhao-yang, LI Xiao-guang,. Cell Cycle Synchronization Methods of Bone Marrow Mesenchymal Stem Cells and Its Effect on Neural Differentiation　[J]. 《Chinese Journal of Rehabilitation Theory and Practice》, 2016, 22(12): 1399-1398.

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骨髓间充质干细胞细胞周期同步化的方法及对向神经细胞分化的影响

Cell Cycle Synchronization Methods of Bone Marrow Mesenchymal Stem Cells and Its Effect on Neural Differentiation

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