一种新生SD大鼠皮质源性神经元的培养方法
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摘要
背景:神经元的体外原代培养在研究神经系统的发育、再生、信号转导机制、神经药理学以及基因表达方面具有极其重要的地位。目的:建立一种操作更加简单且能够得到较高纯度新生SD大鼠皮质神经元原代培养的方法。方法:取1 d新生SD大鼠皮质。传统方法实验组:取整个皮质;改良方法实验组:取SD大鼠表面2.0-3.0 mm处皮质。木瓜蛋白酶消化后离心,制备成单细胞悬液,以1×105/孔的浓度接种至含神经元培养液的24孔培养板进行原代培养。培养3 d时采用免疫细胞化学染色方法,使用神经元特异性标志物Tuj1与MAP-2双标记法鉴定所培养的细胞;采用倒置相差显微镜观察6,24,48,72 h及5,7 d细胞数和突起长度并记录。结果与结论:1所培养的细胞可表达神经元特异性标志物Tuj1与MAP-2,因此所培养细胞为神经元,可用于之后实验;2实验组培养至第3天时神经元的纯度已达到峰值92%,而普通实验组神经元的纯度为51%;32组细胞培养至6 h均已贴壁且长处小突起,培养至第3天时,细胞已初步形成神经网络,培养至5 d时神经网络密集;4研究所用方法简便能够稳定的培养出纯度较高的神经元,可以用于SD大鼠皮质源性神经元的相关实验研究。
BACKGROUND: Primary culture in vitro of neurons plays an important role in the development, regeneration, signal transduction mechanisms, neuropharmacology and gene expressions of the nervous system. OBJECTIVE: To establish a simple method for primary culture of high-purity cortical neurons in neonatal Sprague-Dawley rats.METHODS: Cortical tissues were acquired from neonatal Sprague-Dawley rats born 1 day. In traditional experimental group, the whole cortex was removed; in improved experimental group, the cortical tissues, 2-3 mm thick on the brain surface were removed. Single cell suspensions were prepared after papain digestion and centrifugation and were then seeded onto 24-well culture plates containing neuron solutions for primary culture(1×105 per well). Cells were identified by neuronal specific markers MAP-2 and Tuj1 after 3-day culture. The number of neurons and neurite length were observed under inverted phase contrast microscope and recorded at 6, 24, 48 and 72 hours, 5 and 7 days of culture, resprctively. RESULTS AND CONCLUSION: The cultured cells expressing MAP-2 and Tuj1 were neurons that could be used in the following experiments. The purity of neurons in the improved experimental group was 92% at 3 days, while only 51% in the traditional experimental group. Cells in both two groups had attached to the wall presenting with small processes at 6 hours, and a simple neural network formed at the 3rd day until dense neural networks could be found at the 5th day. To conclude, our culture method herein is simple and convenient, and can be used to produce neurons with high purity, which will be helpful for the experimental studies on cortical neurons from Sprague-Dawley rats.
BACKGROUND: Primary culture in vitro of neurons plays an important role in the development, regeneration, signal transduction mechanisms, neuropharmacology and gene expressions of the nervous system. OBJECTIVE: To establish a simple method for primary culture of high-purity cortical neurons in neonatal Sprague-Dawley rats.METHODS: Cortical tissues were acquired from neonatal Sprague-Dawley rats born 1 day. In traditional experimental group, the whole cortex was removed; in improved experimental group, the cortical tissues, 2-3 mm thick on the brain surface were removed. Single cell suspensions were prepared after papain digestion and centrifugation and were then seeded onto 24-well culture plates containing neuron solutions for primary culture(1×105 per well). Cells were identified by neuronal specific markers MAP-2 and Tuj1 after 3-day culture. The number of neurons and neurite length were observed under inverted phase contrast microscope and recorded at 6, 24, 48 and 72 hours, 5 and 7 days of culture, resprctively. RESULTS AND CONCLUSION: The cultured cells expressing MAP-2 and Tuj1 were neurons that could be used in the following experiments. The purity of neurons in the improved experimental group was 92% at 3 days, while only 51% in the traditional experimental group. Cells in both two groups had attached to the wall presenting with small processes at 6 hours, and a simple neural network formed at the 3rd day until dense neural networks could be found at the 5th day. To conclude, our culture method herein is simple and convenient, and can be used to produce neurons with high purity, which will be helpful for the experimental studies on cortical neurons from Sprague-Dawley rats.
引文
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[14]谢欣梅,庞晓斌,赵艳,等.消栓通络有效成分组对氧糖剥夺损伤原代培养神经元的保护作用[J].药学学报,2014,49(8):1130-1135.
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[21]赵丽,陈瑶,龚丽景,等.乳鼠皮层神经元原代培养改良方法及基本电生理特性研究[J].北京体育大学学报,2012,35(10):42-45.
[22]马万,袁文俊,焦英甫,等.一种稳定高效的新生大鼠皮质神经元原代培养方法[J].神经解剖学杂志,2015,31(4):510-514.
[23]杨传豪,赵冬,刘祺,等.新生大鼠皮层神经元体外无血清原代培养[J].重庆医学,2014,43(29):3901-3906.
[24]张明,郭艺,蒋莉,等.无血清原代培养大鼠海马神经元的形态与膜电位[J].细胞生物学杂志,2007,29(1)):131-134.
[25]郭琴,毛萌,罗小丽,等.神经元原代培养方法的改进[J].华西医学,2007,22(3):566-567.
[26]潘永英,彭书崚,赵一凡,等.大鼠海马神经元的原代无血清培养及鉴定[J].实用医学杂志,2006,22(7):758-759.
[27]邓莉,代荣阳,廖彦生,等.新生大鼠大脑皮层神经元原代培养与鉴定[J].现代医药卫生,2011 27(5):646-645.
[28]郭琴,毛萌,罗小丽,等.神经元原代培养方法的改进[J].华西医学,2007,22(3):566-567.
[29]宋海岩,王志勇,李娜娜.新生大鼠大脑皮质神经元的原代培养及其鉴定[J].实用儿科临床杂志,2012,27(2):129-131.
[30]梅莉,路浩,刘宗平,等.新生大鼠大脑皮质神经元原代培养方法的建立及其活性鉴定[J].中国兽医科学,2007,37(3):264-268.
[31]宋岳,洪庆涛,唐一鹏.阿糖胞苷对原代培养的大鼠大脑皮层神经元的影响[J].解剖学杂志,2004,27(6):696-699.
[2]司徒镇强,吴军正.细胞培养[M].北京:世界图书出版公司,2007:119-122.
[3]鄂征.组织培养技术及其在医学研究中的应用[M].北京:中国协和医科大学出版社,2004:65-69.
[4]Robert F,Cloix JF,Hevor T.Ultrastructural characterization of rat neurons in primary culture.Neuroscience.2012;200:248-260.
[5]张玮,唐卉凌,王筠,等.大鼠大脑皮质神经元原代培养不同培养体系及纯化方法的比较研究[J].现代生物医学进展,2010,10(6):1043-1046.
[6]王圆圆,张文斌,龚晓兵,等.大鼠海马神经元neurobasal无血清的原代培养方法[J].暨南大学学报:医学版,2008,29(6):547-551.
[7]姜骏,符岳,毛小英,等.机械吹打法大鼠海马神经元原代培养的研究[J].岭南急诊医学杂志,2013,18(3):163-165
[8]姜茜,姜玉武,王静敏,等.一种改进的大鼠皮层神经元原代培养方法及其性质鉴定[J].北京大学学报:医学版,2009,41(2):212-216.
[9]周国凤,汤京龙,周亮.一种改进的大脑皮层神经元原代培养方法的研究[J].药物分析杂志,2011,31(2):299-301.
[10]Ravishankar S,Ashraf QM,Fritz K,et al.Expression of Bax and Bcl-2 proteins during hypoxia in cerebral cortical neuronal nuclei of newborn piglets:effect of administration of magnesium sulfate.Brain Res.2001;901(1):23-29.
[11]Xiang K,Earl D,Dwyer T,et al.Hypoxia enhances high voltage activated calcium currents in rat primary cortical neurons via calcineurin.Epilepsy Res.2012;99(3):293-305.
[12]莽靖,李昕华,阎世军,等.大鼠皮层神经元原代培养、鉴定及氧糖剥夺模型的建立[J].中国老年学杂志,2010,30(6):790-791.
[13]张琛,叶瑞东,韩军良,等.人参皂甙Rd对皮层神经元兴奋性毒性损伤后胞内游离钙的影响[J].神经解剖学杂志,2010,26(2):112-118.
[14]谢欣梅,庞晓斌,赵艳,等.消栓通络有效成分组对氧糖剥夺损伤原代培养神经元的保护作用[J].药学学报,2014,49(8):1130-1135.
[15]左红艳,王德文,彭瑞云,等.电磁辐射对原代培养海马神经元的损伤效应及其机制探讨[J].生物物理学报,2007,23(1):47-53.
[16]李玮,石晋丽,李琴,等.甘松新酮对缺糖缺氧损伤原代培养神经元的保护作用[J].药学学报,2013,48(9):1422-1429.
[17]田凤艳,宁琴,罗小平.大鼠原代皮质神经元无血清培养的影响因素及优化培养方法[J].实用儿科临床杂志,2010,25(14):1094-1097.
[18]廖慧丹,龙玲玲,闫杰.三种皮质神经元原代培养方法的改良及对比[J].2015,23(4):410-414.
[19]余华荣,杨贵忠,杨俊卿,等.大鼠皮层神经元原代培养方法的改进[J].重庆医科大学学报,2007,32(4):392-394.
[20]曹珂,刘检,苗露阳,等.皮质神经元B27原代培养及MAP2鉴定[J].沈阳药科大学学报,2013,30(1):40-43.
[21]赵丽,陈瑶,龚丽景,等.乳鼠皮层神经元原代培养改良方法及基本电生理特性研究[J].北京体育大学学报,2012,35(10):42-45.
[22]马万,袁文俊,焦英甫,等.一种稳定高效的新生大鼠皮质神经元原代培养方法[J].神经解剖学杂志,2015,31(4):510-514.
[23]杨传豪,赵冬,刘祺,等.新生大鼠皮层神经元体外无血清原代培养[J].重庆医学,2014,43(29):3901-3906.
[24]张明,郭艺,蒋莉,等.无血清原代培养大鼠海马神经元的形态与膜电位[J].细胞生物学杂志,2007,29(1)):131-134.
[25]郭琴,毛萌,罗小丽,等.神经元原代培养方法的改进[J].华西医学,2007,22(3):566-567.
[26]潘永英,彭书崚,赵一凡,等.大鼠海马神经元的原代无血清培养及鉴定[J].实用医学杂志,2006,22(7):758-759.
[27]邓莉,代荣阳,廖彦生,等.新生大鼠大脑皮层神经元原代培养与鉴定[J].现代医药卫生,2011 27(5):646-645.
[28]郭琴,毛萌,罗小丽,等.神经元原代培养方法的改进[J].华西医学,2007,22(3):566-567.
[29]宋海岩,王志勇,李娜娜.新生大鼠大脑皮质神经元的原代培养及其鉴定[J].实用儿科临床杂志,2012,27(2):129-131.
[30]梅莉,路浩,刘宗平,等.新生大鼠大脑皮质神经元原代培养方法的建立及其活性鉴定[J].中国兽医科学,2007,37(3):264-268.
[31]宋岳,洪庆涛,唐一鹏.阿糖胞苷对原代培养的大鼠大脑皮层神经元的影响[J].解剖学杂志,2004,27(6):696-699.