中国组织工程研究

中国组织工程研究 ›› 2012, Vol. 16 ›› Issue (49): 9241-9246.doi: 10.3969/j.issn.2095-4344.2012.49.022

• 干细胞培养与分化 stem cell culture and differentiation • 上一篇    下一篇

神经干细胞体外衰老模型的构建及相关生物学特点

彭 彬1,2，王朝丽2，冯 丽2，王亚平1   

  1. 1重庆医科大学干细胞与组织工程研究室，重庆市   400016；2川北医学院细胞化学研究室，四川省南充市 637007
  • 收稿日期:2012-03-07 修回日期:2012-04-09 出版日期:2012-12-02 发布日期:2012-12-02
  • 通讯作者: 王亚平，教授，博士生导师，重庆医科大学干细胞与组织工程研究室，重庆市 400016 ypwangcq@yahoo.cn
  • 作者简介:彭彬☆，女，1972年生，四川省南充市人，汉族，2011年重庆医科大学毕业，博士，副教授，主要从事神经干细胞衰老生物学研究。 binpeng0817@foxmail.com
  • 基金资助:

    四川省教育厅项目(10ZA176)

In vitro establishment of a neural stem cell aging model and investigation of its biological
characteristics

Peng Bin1, 2, Wang Chao-li2, Feng Li2, Wang Ya-ping1   

  1. 1Laboratory of Stem Cell and Tissue Engineering, Chongqing Medical University, Chongqing 400016, China; 2Cytochemical Research Laboratory, North Sichuan Medical College, Nanchong 637007, Sichuan Province, China
  • Received:2012-03-07 Revised:2012-04-09 Online:2012-12-02 Published:2012-12-02
  • Contact: , Professor, Doctoral supervisor, Laboratory of Stem Cell and Tissue Engineering, Chongqing Medical University, Chongqing 400016, China ypwangcq@yahoo.cn
  • About author:Peng Bin☆, Doctor, Associate professor, Laboratory of Stem Cell and Tissue Engineering, Chongqing Medical University, Chongqing 400016, China, Cytochemical Research Laboratory, North Sichuan Medical College, Nanchong 637007, Sichuan Province, China binpeng0817@foxmail.com
  • Supported by:

    Project of Education of Sichuan Province, No. 10ZA176*

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摘要：

背景：干细胞衰老主要表现为其增殖、分化能力下降，端粒长度缩短及端粒酶活性增强以及衰老相关基因及蛋白的表达上调等。
目的：建立神经干细胞体外衰老模型，探讨其衰老的相关生物学特点及调控机制。
方法：将从新生SD大鼠海马组织中分离纯化的第3代神经干细胞在37 ℃、体积分数5% CO2和神经干细胞完全培养基中培养2 h为对照组，在对照组基础上加入终浓度为100 μmol/L 三丁基过氧化氢培养2 h构建神经干细胞体外衰老模型为衰老模型组。
结果与结论：与对照组比较，三丁基过氧化氢作用2 h，倒置显微镜下见衰老模型组神经干细胞增殖形成的神经球数量、体积以及由其分化形成的细胞数量均显著下降；MTT吸光度值显著下降26%，神经干细胞增殖形成的神经球数量下降48%；神经干细胞分化形成的神经元的数密度显著下降61%；衰老相关β-半乳糖苷酶阳性神经球百分比显著升高了19倍；p16INK4a、p21Cip1/Waf1 mRNA的表达水平分别显著升高了137%和68%。以上结果表明，三丁基过氧化氢能构建神经干细胞体外衰老模型，p16INK4、p21Cip1/Waf1可能通过p16/RB和p19ARF/p53/ p21cip1通路调控三丁基过氧化氢诱导神经干细胞衰老。

关键词: 神经干细胞, 衰老, 体外模型, 三丁基过氧化氢, 大鼠

Abstract:

BACKGROUND: Stem cell aging is mainly manifested by the decreased proliferation and differentiation ability, shortened telomere length, increased telomerase activity and increased expression of aging-related gene and protein.  
OBJECTIVE: To establish a neural stem cell aging model in vitro and to explore the aging-related biological characteristics and mechanisms.
METHODS: The third generation of neural stem cells were isolated and purified from the hippocampus of newborn Sprague-Dawley rats, and the cells were cultured in the medium containing 5% CO2 and neural stem cells under 37 ℃ for 2 hours (control group), then 100 mol/L tert-butyl hydroperoxide was added to the cells for 2 hours to establish the neural stem cell aging model in vitro (model group).
RESULTS AND CONCLUSION: Compared with the control group, after being treated with tert-butyl hydroperoxide for 2 hours, the number and volume of neurospheres forming by proliferated neural stem cells significantly decreased and the number of differentiated cells was significantly decreased in the model group; the absorbance was detected by MTT, the quantity of neurospheres and the numerical density of neurons were significantly decreased by 26%, 48% and 61%, respectively; the percentage of senescence-associated β-galactosidase glucosidase-positive neurospheres was increased by 19 times, and the expression levels of p16INK4a and p21Cip1/Waf1 mRNA were significantly increased by 137% and 68%. These results suggest that tert-butyl hydroperoxide can be used to establish the aging model in vitro. p16INK4 and p21Cip1/Waf1 may play a key role in regulating aging process induced by tert-butyl hydroperoxide through signal transduction pathway of p16/Rb and p19ARF/ p53/ p21cip1.

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