2种原代培养方法对新生大鼠大脑皮质神经元生物学特性的影响
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摘要
为明确不同培养方法对新生大鼠大脑皮质神经元成熟时间、形态特征、纯度及活力等生物学特性的影响,采用DMEM培养基加Neurobasal无血清培养基法或Neurobasal无血清培养基法原代培养新生24h内SD大鼠大脑皮质神经元,倒置显微镜下观察细胞形态,MTT法检测细胞活力,免疫荧光细胞化学染色法检测神经元纯度及活性.结果显示,2种方法培养的神经元形态差异无统计学意义;但与DMEM培养基加Neurobasal无血清培养基法相比,Neurobasal无血清培养基法培养的神经元成熟更早,数目更多,纯度更高,活力更强(p<0.05).结果提示,原代培养的大脑皮质神经元部分生物学特性受培养方法直接影响;Neurobasal无血清培养基法所得神经元纯度与活性较高,这为实验目的导向的神经元原代培养方法选择提供了借鉴.
To clarify the effects of two primary culture methods on the maturation time span,morphological feature,purity,viability and activity of cerebral cortical neurons,cortical neurons were obtained from newly born SD rats and plated in DMEM + neurobasal and neurobasal nutrient media,respectively.The neuronal morphology was visualized using an inverted contrast microscope,MTT assay was used to evaluate the cellular viability,and immunofluorescence staining was employed to identify the purity and activity of the neurons.No obvious difference between the two methods was observed in their effect on neuronal morphology.However,cellular viability,purity and activity of the neurons in the neurobasal nutrient medium were significantly higher than those of the other method(p<0.05).Moreover,the number of cells was greater and the maturation time span was shorter in the neurobasal nutrient medium.These results showed that the biological characteristics of the cerebral cortical neurons in primary culture were directly affected by the culture method.The neurobasal serum-free culture program had higher purity and activity of neurons,which provided a reference for the selection of primary culture methods for neuronal priming.
To clarify the effects of two primary culture methods on the maturation time span,morphological feature,purity,viability and activity of cerebral cortical neurons,cortical neurons were obtained from newly born SD rats and plated in DMEM + neurobasal and neurobasal nutrient media,respectively.The neuronal morphology was visualized using an inverted contrast microscope,MTT assay was used to evaluate the cellular viability,and immunofluorescence staining was employed to identify the purity and activity of the neurons.No obvious difference between the two methods was observed in their effect on neuronal morphology.However,cellular viability,purity and activity of the neurons in the neurobasal nutrient medium were significantly higher than those of the other method(p<0.05).Moreover,the number of cells was greater and the maturation time span was shorter in the neurobasal nutrient medium.These results showed that the biological characteristics of the cerebral cortical neurons in primary culture were directly affected by the culture method.The neurobasal serum-free culture program had higher purity and activity of neurons,which provided a reference for the selection of primary culture methods for neuronal priming.
引文
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[20]李昂,晏芳,李振林,等.探讨阿糖胞苷在原代大鼠海马神经元培养中的纯化方法[J].中国临床解剖学杂志,2015,33(6):681-684,689.
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[23]PIALI L,HAMMEL P,UHEREK C,et al.CD31/PECAM-1Is a Ligand forαvβ3Integrin Involved in Adhesion of Leukocytes to Endothelium[J].The Journal of Cell Biology,1995,130(2):451-460.
[24]LACHYANKAR MB1,SULTANA N,SCHONHOFF C M,et al.A Role for Nuclear PTEN in Neuronal Differentiation[J].J Neurosci,2000,20(4):1404-1413.
[2]KAECH S,BANKER G.Culturing Hippoeampal Neurons[J].Nat Protoc,2006,1(5):2406-2425.
[3]林清,王玮.大鼠大脑皮质神经元的原代培养[J].福建医科大学学报,2008,42(2):152-154,160.
[4]王涛.梓醇对“神经血管单元”保护作用的研究[D].重庆:西南大学,2015.
[5]BREWER G J,TORRICELLI J R,EVEGE E K,et al.Optimized Survival of Hippocampal Neurons in B27Supplemented Neuroba1.A New Serum-Free Medium Combination[J].J Neurosci Res,1993,35(5):567-576.
[6]周国凤,汤京龙,周亮,等.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.
[7]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.
[8]PONGRAC J L,RYLETT R J.Optimization of Serum-Free Culture Conditions for Growth of Embryonic Rat Cholinergic Basal Forebrain Neurons[J].Journal of Neuroscience Methods,1998,84(1-2):69-76.
[9]BREWER G J.Serum-Free B27 Neurobasal Medium Supports Differentiated Growth of Neurons From the Striatum,Substantia Nigra,Septum,Cerebral Cortex,Cerebellum,and Dentate Gyrus[J].Journal of Neuroscience Research,1995,42(5):674-683.
[10]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.
[11]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.
[12]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].中国实验动物学报,2015,23(4):410-414.
[13]郭明星,陈浩宇,梁璐,等.新生大鼠海马神经元原代培养方法的优化与鉴定[J].湖北科技学院学报(医学版),2016,30(4):284-287.
[14]吴秀云,凌勇,张殿隆,等.一种改良大鼠海马神经元原代培养的方法[J].青岛大学医学院学报,2016,52(5):519-521.
[15]张涛,胡怀强,王旭辉,等.一种高效简便的原代神经元培养方法[J].神经损伤与功能重建,2016,11(6):471-472,475.
[16]汤军,丁嵩涛,刘莉,等.柳氮磺胺吡啶对胶原诱导关节炎Th17/Treg细胞的影响[J].西南大学学报(自然科学版),2016,38(5):66-70.
[17]张学平,王高华,王惠玲,等.海马神经元原代培养与纯度鉴定方法的优化[J].武汉大学学报(医学版),2013,34(5):703-706,710.
[18]PARK K K,LIU K,HU Y,et al.Promoting Axon Regeneration in the Adult CNS by Modulation of the PTEN/mTOR Pathway[J].Science,2008,322(5903):963-966.
[19]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.
[20]李昂,晏芳,李振林,等.探讨阿糖胞苷在原代大鼠海马神经元培养中的纯化方法[J].中国临床解剖学杂志,2015,33(6):681-684,689.
[21]李少兵,韩卉.神经营养因子与周围神经再生[J].解剖科学进展,2001,7(3):265-270.
[22]GAO Yu-hua,LI Xiang-chen,ZHENG Dong,et al.Isolation of a Pluripotent Neural Stem Cell from the Embryonic Bovine Brain[J].Int J Mol Sci,2015,16(3):5990-5999.
[23]PIALI L,HAMMEL P,UHEREK C,et al.CD31/PECAM-1Is a Ligand forαvβ3Integrin Involved in Adhesion of Leukocytes to Endothelium[J].The Journal of Cell Biology,1995,130(2):451-460.
[24]LACHYANKAR MB1,SULTANA N,SCHONHOFF C M,et al.A Role for Nuclear PTEN in Neuronal Differentiation[J].J Neurosci,2000,20(4):1404-1413.