生物医学  >> Vol. 1 No. 2 (October 2011)

新生大鼠神经干细胞的体外培养与冷冻复苏
Cryopreservation and Culture of Neural Stem Cells Isolated from Postnatal Rat

DOI: 10.12677/hjbm.2011.12005, PDF, HTML, XML, 下载: 3,473  浏览: 14,694  科研立项经费支持

作者: 张静, 康现江, 周辰, 张平, 穆淑梅, 张晗

关键词: 神经干细胞新生大鼠体外培养冻存复苏
Neural Stem Cells; Postnatal Rat; Culture in Vitro; Cryopreservation; Resuscitation

摘要: 目的:分离培养新生SD大鼠神经干细胞(NSCs),观察应用不同浓度的二甲基亚砜(DMSO)作为冷冻保护剂冷冻保存不同时间的神经干细胞复苏后,其活力及生物学特性的鉴定。方法:在含细胞因子表皮生长因子及碱性成纤维细胞生长因子的无血清培养液中培养新生大鼠海马神经干细胞。经传代扩增后,培养于含有0、5%、10%、15%、20%不同浓度的二甲基亚砜和胎牛血清的神经干细胞冻存液中,在–80℃冰箱中分别冻存1周、2周及一个月后,复苏培养并鉴定,然后对其复苏率进行检测。结果:新生大鼠海马神经干细胞经冷冻复苏后,大多数细胞生长良好并形成新的克隆球。台盼蓝染色检测冻存1周、2周及一个月后的复苏率,含有10%二甲基亚砜的复苏率最高且具有显著性差异(P < 0.05),不同冻存时间的复苏率分别为54.00 ± 1.73,59.00 ± 1.16,58.00 ± 2.08。结论:神经干细胞能在体外适宜的条件下进行培养及冻存、复苏,并且复苏后不影响其原有的生物学特性。
Abstract: Objective: to isolate and culture neural stem cells from postnatal rat and to observe the viability and biological property of these cells after cryopreservation. Methods: the neural stem cells isolated from hippocampus of the postnatal rat were cultured in culture solution without blood serum. After amplification, the cells were cultured in different concentration of freeze-stored liquid which contained 0、5%、10%、15%、20%DMSO and fetal calf serum, then stored in refrigerator of 80 centigrade below zero one week, two weeks and one month respectively. Through resuscitation training and testing, then we detected the recovery rate of the NSCs. Results: after cryopreservation and culture of neural stem cells isolated from postnatal rat, most of them were growth well and formed into the new cell clones, then the recovery rate of the cells were de-tected with Trypan Blue. There was significant difference in 10% DMSO and it has the highest recovery rate,which is 54.00 ± 1.73, 59.00 ± 1.16, 58.00 ± 2.08. Conclusion: the neural stem cells derived from postnatal rat were able to culture, cryopreservation and resuscitation, which did not change their biological properties.

文章引用: 张静, 康现江, 周辰, 张平, 穆淑梅, 张晗. 新生大鼠神经干细胞的体外培养与冷冻复苏[J]. 生物医学, 2011, 1(2): 25-33. http://dx.doi.org/10.12677/hjbm.2011.12005

参考文献

[1] 黄毓, 覃甲仁. 神经干细胞的研究进展[J]. 广西医科大学学报, 2004, 21(2): 308-310.
[2] B. A.Reynolds, S. Weiss. Generation of neurons and astrocytes from isolated cells of adult mammalian central nervous system. Science, 1992, 255(5052): 1707-1710.
[3] L. J. Richards, T. J. Kilpatrick and P. F. Bartlett. De novo genera-tion of neural cells from the adult mouse brain. Proceeding of the National Academy Sciences of the United States of America, 1992, 89(18): 8591-8595.
[4] P. S. Eriksson, E. Perfilieva, T. Bjork-Eriksson, et al. Neuro-genesis in the adult human hippocampus. National Medecine, 1998, 4(11): 1313-1317.
[5] C. B. Johansson, M. Svensson, L. Wallstedt, et al. Neural stem cells in the adult human brain. Experimental Cell Research, 1999, 253(2): 733-736.
[6] S. Temple. The development of neural stem cells. Nature, 2001, 414(6859): 112-117.
[7] 罗小丹, 沈岳飞. 神经干细胞定向诱导分化为多巴胺能神经元的研究进展[J]. 神经解剖学杂志, 2009, 25(4): 469-473.
[8] V. G. Kukekvo, E. D. Laywell, O. Suslov, et al. Multipotent stem cells with similar progerties arise from two neurogenic regions of adult human brain. Experimental Neurology, 1999, 156(2): 333-344.
[9] B. A. Reynolds, A. Weiss. Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous sys-tem. Science, 1992, 255(5052): 1707-1710.
[10] A. A. Davis, S. Temple. A self-renewing multipotential stem cells in embryonic rat cerebral cortex. Nature, 1994, 372(6503): 263-266.
[11] J. Dietrich, M. C. Easterday. Developing concepts in neural stem cells. Trends in Neurosciences, 2002, 25(3): 129-131.
[12] M. Nakamura, Y. Toyama and H. Okano. Transplantation of neural stem cells for spinal cordinjury. Shinkeigaku, 2005, 45(11): 874-876.
[13] 陈凯, 康现江, 张平等. 大鼠海马神经干细胞的分离培养与免疫荧光鉴定[J]. 医学研究教育, 2010, 27(2): 1-3.
[14] Z. Q. Shu, X. J. Kang, H. H. Chen, et al. Development of a reliable low-cost controlled cooling rate instruments for the cryopreserva-tion of hematopoietic stem cells. Cytotherapy, 2010, 12(2): 161- 169.
[15] R. Mckay. Stem cells in the central nervous system. Science, 1997, 276(5309): 66-71.
[16] Gaiano N, Fishell G. Transplantation as a tool to study progeni-tors within the vertebrate nervous system. Neurobiol, 1998, 36(2): 152-161.
[17] Y. Ogawa, K. Sawamoto, T. Miyata, et al. Transplantation of in vitro-expanded fetal neural progenitor cells results in neurogene-sis and functional recovery after spinal cord contusion injury in adult rats. Journal of Neuroscience Research, 2002, 69(6): 925- 933.
[18] P. Akerud, J. M. Canals, E. Y. Snyder, et al. Neuroprotection through delivery of glial cell linederived neurotrophic factor by neural stem cells in a mouse model of Parkinson’s disease. The Journal of Neuroscience, 2001, 21(20): 8108-8118.
[19] 周燕琼, 刘小辉, 刘幸平等. 人肝癌细胞冷冻保存方法研究[J]. 世界华人消化杂志, 2000, 8(6): 714-715.
[20] X. M. Zhou, X. J. Kang, Z. Q. Shu, et al. Cryopreservation of peripheral blood stem cells using a box-in-box cooling device. Biopreservation and Boibanking, 2009, 7(2): 107-114.
[21] A. Gritti, E. A. Parati, L. Cova, et al. Multipotential stem cells from the Adultmouse brain proliferate and self-renew in response to basic fibroblast growth factor. The Journal of Neuroscience, 1996, 16(3): 1091-1100.
[22] N. Uchida, D. W. Buck, D. He, et al. Direct isolation of human central nervous system stem cells. Proceeding of the National Academy Sciences of the United States of America, 2000, 97(26): 14720-14725.
[23] 胡钧涛, 涂汉军, 张力等. 大鼠胚胎神经干细胞两种不同分离、传代方法的比较[J]. 中国神经精神疾病杂志, 2006, 32(2): 164-166.
[24] W. W. Tornford, G. P. Duff and H. J. Mankin. Experimental freezepreservation of chondrocytes. Clinical Orthopaedics & Related Research, 1985, 7(197): 11-14.