康杨婷,王丽琨,伍国锋
KANG Yangting,WANG Likun,WU Guofeng

• 1:贵州医科大学
• 2:贵州医科大学附院急诊神经科

摘要(Abstract)：

目的:优化SD乳鼠海马神经元的提取方法。方法:选用出生24 h内的SD乳鼠,分离双侧海马组织,分别用胰蛋白酶稀释法和未稀释法消化后获得细胞悬液;培养细胞,在培养第7天采用神经元特异性烯醇化酶(NSE)免疫荧光染色鉴定神经元数量及纯度。结果:胰酶稀释组提取得到的海马神经元结构特征明显,能形成典型的神经细胞网络,90%以上海马神经细胞呈NSE阳性。结论:用稀释胰蛋白酶消化海马组织并配合特定培养条件可获得生长状态良好、纯度高的原代海马神经细胞。
Objective:To optimize the method for isolating primary hippocampal neurons from newborn mice.Methods:In SD rats newborn within 24 hours of birth,bilateral hippocampus tissues were isolated and digested with 0.25% trypsin and undiluted trypsin(0.125%) to obtain two types of cell suspension,respectively.Cells were cultured.On day 7 of culture,cells were immunofluorescently stained for detecting neuron-specific enolase(NSE) to identify the number and purity of neurons.Results:The isolated hippocampal neurons using 0.125% trypsin had obvious structural features and could form a typical neural cell network.Moreover,over 90% of hippocampal neurons were positive for NSE.Conclusion:Isolated primary hippocampal neurons using 0.125% trypsin had higher purity than using 0.25% trypsin and grew well under proper culture condition.

关键词(KeyWords)： 胰酶;稀释技术;神经元,海马;细胞培养;鉴定
trypsin;dilution;neurons,hippocampal;cell culture;identification

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金(81760245/H0913,81560222/H0913);; 贵州省优秀教育科技人才省长专项资金(1065-09);; 贵州省高层次人才科研特助经费(TZJF-2010-054)

作者(Author): 康杨婷,王丽琨,伍国锋
KANG Yangting,WANG Likun,WU Guofeng

DOI: 10.19367/j.cnki.1000-2707.2019.01.011

参考文献(References)：

• [1]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,2013,34(5):703-706.
• [2]KIM S,DEDE A J,HOPKINS R O,et al.Memory,scene construction,and the human hippocampus[J].Proc Nat Acad Sci USA,2015,112(15):4767-4770.
• [3]FASICK V,SPENGLER R N,SAMANKAN S,et al.The hippocampus and TNF:Common links between chronic pain and depression[J].Neurosci Biobehav Rev,2015,53:139-159.
• [4]CIRNECI D,SILAGHI-DUMITRESCU R.Learning tasks as a possible treatment for DNA lesions induced by oxidative stress in hippocampal neurons[J].Neural Regen Res,2013,8(32):3063-3070.
• [5]CHRISTIAN K M,SONG H,MING G L.Functions and dysfunctions of adult hippocampal neurogenesis[J].Annu Rev Neurosci,2014,37:243-262.
• [6]赵秀鹤,迟兆富,尚伟,等.新生大鼠海马神经元的体外原代培养[J].基础医学与临床,2007,27(3):329-332.
• [7]陈江瑛,闰振文,张素平,等.氯沙坦对离体培养大鼠海马神经元缺糖缺氧损伤的保护作用[J].中国临床药理学与治疗学,2014,19(9):984-987.
• [8]仁德芳,付裕,王洪连,等.乳鼠海马神经元的分离和原代培养[J].细胞与分子免疫学杂志,2017,33(5):660-663.
• [9]黄孝闻,王绪平,寿旦.海马神经元细胞模型及其应用的研究进展[J].中华中医药学刊,2015(11):2730.
• [10]黄立宁.氯胺酮对发育期海马神经元凋亡的影响及机制研究[D].石家庄:河北医科大学,2012.
• [11]SEIBENHENER M L,WOOTEN M W.Isolation and culture of hippocampal neurons from prenatal mice[J].JVis Exp,2012(65):e3634.
• [12]郭慧,俞丹,周晖,等.新生大鼠海马神经元细胞体外培养方法改良研究[J].中华妇幼临床医学杂志,2016,12(1):24-28.
• [13]谢丽,赵树进,严华成,等.一种简单高效的新生鼠海马神经元原代培养方法[J].广州中医药大学学报,2016,33(4):570-573.
• [14]BELLOZI P M,LIMA I V,DORIA J G,et al.Neuroprotective effects of the anticancer drug NVP-BEZ235(dactolisib)on amyloid-β1induced neurotoxicity and memory impairment[J].Sci Rep,2016,6:25226.
• [15]HOSHINO M,TSUJIMOTO T,YAMAZOE S,et al.Adhesamine,a new synthetic molecule,accelerates differentiation and prolongs survival of primary cultured mouse hippocampal neurons[J].Biochem J,2010,427(2):297-304.
• [16]LU Z,PIECHOWICZ M,QIU S,et al.A simplified method for ultra-low density,long-term primary hippocampal neuron culture[J].Vis Exp,2016(109):e53797.
• [17]GAO S,YU Y,MA Z Y,et al.NMDAR-mediated hippocampal neuronal death is exacerbated by activities of ASIC1a[J].Neurotox Res,2015,28(2):122-137.
• [18]PANAS D,AMIN H,MACCIONE A,et al.Sloppiness in spontaneously active neuronal networks[J].J Neurosci,2015,35(22):8480-8492.
• [19]徐祖才,徐平,张骏,等.新生大鼠海马神经元原代培养及膜片钳全细胞记录[J].重庆医学,2012,41(31):3241-3244.
• [20]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.
• [21]HU W Y,HE Z Y,YANG L J,et al.The Ca 2+channel inhibitor 2-APB reversesβ-amyloid-induced LTP deficit in hippocampus by blocking BAX and caspase-3 hyperactivation[J].Br J Pharmacol,2015,172(9):2273-2285.
• [22]刘检,王宇红,徐雅岚,等.改良的胎鼠海马神经元原代培养及模拟糖尿病并发抑郁环境对其的损伤作用[J].神经解剖学杂志,2016,32(4):459-465.
• [23]郭慧,俞丹,周晖,等.新生大鼠海马神经元细胞体外培养方法改良研究[J].中华妇幼临床医学杂志,2016,12(1):24-28.
• [24]张馥镇.丙泊酚及右美托咪定对体外培养海马神经元的影响及机制研究[D].石家庄:河北医科大学,2015.

文章评论(Comment)：