大鼠海马神经元及星形胶质细胞的体外培养  

Culture Method of Rat Fetal Hippocampal Neurons and Astrocytes

作　　者：张楠[1] 熊文雯 邢媛 张炜[1] ZHANG Nan;XIONG Wen-wen;XING Yuan;ZHANG Wei(Institute of Chinese Integrative Medicine,Hebei Medical University,Shijiazhuang,050017,China;The Department of Maternity,Guangdong Health Center for Women and Children,Guangzhou,510000,China;Hebei North University,Zhangjiakou,075000,China)

机构地区：[1]中西医结合研究所,河北医科大学,石家庄050017 [2]广东省妇幼保健院妇科,广州510000 [3]河北北方学院,张家口075000

出　　处：《神经药理学报》2017年第1期24-28,共5页Acta Neuropharmacologica

基　　金：国家自然科学基金资助项目(No.31200808;81573416)

摘　　要：目的:改进大鼠海马神经元、星形胶质细胞及两者混合状态的体外培养方法,获得高纯度的海马神经细胞。方法:选取孕期18~20 d的SD大鼠胎鼠作为原代海马细胞培养的材料来源。断头后于冰板上剥离双侧海马,去除表面血管及薄膜,利用眼科剪分离为小块。随后使用冷平衡盐溶液洗涤3次,将海马组织碎块移入酶消化液中。本方法选择Accutase和0.1%DNase作为酶消化液,37℃消化15 min,期间轻柔振动。使用冷平衡盐溶液洗涤3次,终止消化。0.1%DNase加入Neurobasal和B-27的混合液对消化后的海马组织碎块进行轻柔吹打,获得单细胞悬液。将悬液过200目筛,进行细胞计数。将过筛的单细胞悬液移入DMEM培养基(DMEM+10%胎牛血清)中,按照每平方厘米2.5×104的细胞密度进行接种。经PDL孵育的塑料片置于培养基底部。细胞于37℃、5%CO2培养箱内培养。4 h后,依据所需细胞种类的不同,更换相应培养基。海马神经细胞于培养后7~10 d可用于实验。结果:通过本方法可得到高纯度的大鼠海马神经细胞,且根据实验需求,通过更换培养基,可得到纯神经元、纯胶质细胞及两者混合的三种培养状态。培养的神经元及星形胶质细胞纯度均>98%。结论:该文在传统海马神经细胞体外培养方法基础上,进一步降低了实验操作难度,方法稳定有效。Objective:To develop an improved culture method of neurons and astrocytes from hippocampus of SD rat fetuses in order to aquire high purity hippocampal cultures.Methods:The cultures of primary hippocampal neurons and astrocytes were prepared from SD rat fetuses at gestation day18~20.After decapitation,the rat embryonic hippocampal tissue was taken out,sepatated and cutted into cubes quickly on ice.Then hippocampal cubes were transferred to the dissociation enzyme slolution after being washed in HBSS for three times.We chose Accutase and0.1%Dnase as the digestion enzymes.The hippocampal tissue was incubated at37℃for15min with gentle shaking.Tissue digestion was stopped by being washed with HBSS for three times.After digestion,the tissue was gently triturated with triturating solution containing Neurobasal,B-27and0.1%DNase.The cell suspension was passed through sterile nylon gauze(80mm).The hippocampal cell suspension was plated in24-well plates coated with poly-D-lysine at a density of2.5×104cells·cm-2.The cultures were maintained at37℃in a humidified atmosphere of5%CO2in DMEM medium supplemented with10%Fetal Bovine Serum.After4h of initial plating,the medium was replaced with different medium according to targeting culture status(pure neurons,pure astrocytes or mixed culture).After7~10days in culture,the rat hippocampal cells could be used in follow-up experiments.Results:With this method,we aquired high purity hippocampal culture from rats.By changing different medium,we aquired different culture status,pure culture of neurons or astrocytes,or mixed culture of both cell types.The purity of pure culture cells was higher than98%(both neurons and astrocytes).Conclusion:We developed an improved culture method based on traditional cultivation.This method has lower difficulty in operation and higher stabili ty.

关 键 词：细胞培养 海马 神经元 星形胶质细胞 

分 类 号：R965.2[医药卫生—药理学]