建立一种简便的大鼠背根神经节神经元原代培养方法的建立  

Setting up a simple method to obtain primary cultured dorsal root ganglion neurons in the rat

作　　者：郭琼[1] 李甜[1] 冯树梅[1] 王磊[2] 

机构地区：[1]新疆医科大学基础医学院组织学与胚胎学教研室,乌鲁木齐830011 [2]新疆乌鲁木齐兵团医院创伤科,乌鲁木齐830002

出　　处：《神经解剖学杂志》2014年第2期167-172,共6页Chinese Journal of Neuroanatomy

基　　金：新疆医科大学校内支撑学科项目-人体解剖与组织胚胎学阶段性成果(XYDXK50780307)

摘　　要：目的:建立一种简便易行地获取大鼠背根神经节(dorsal root ganglion,DRG)神经元原代培养的方法。方法:窒息法处死大鼠,打开胸腔经心脏放血后,从背部取出一段脊柱。将脊柱稍加修剪后,从中轴线处打开脊柱,暴露脊髓,在其两侧找到椎间孔内的为DRG,用器械离断神经纤维,取出DRG。Ⅰ胶原酶联合Trypsin-EDTA先消化DRG,再用吹法DRG以获得单个神经元;用TNB-100 basal Medium的复合培养液在经过0.1 mg/ml PolyD-lysine hydrobromide和0.01 mg/ml lamina包被的培养皿内,于5%CO2,37℃孵箱内培养48 h。免疫组织化学方法检测神经元内标志物BetaⅢTubulin的表达,用Alexa flour 488标记的二抗进行显色,倒置荧光显微镜下观察。结果:获得了单层贴壁的神经元,其胞体形状多样,突起生长良好,细胞之间正在逐步形成网络连接;免疫荧光染色显示,细胞的胞体和突起中均可高表达BetaⅢTubulin,且荧光染色较强。结论:运用该方法可以简便地从大鼠DRG内获得单层贴壁的原代神经元。Objective： Setting up a simple method to obtain primary culture neurons of dorsal root ganglion （DRG） of rat. Methods： Kill the rat with asphyxia way, open the chest and let the blood flow out by cuting heart. We get the spine from the rat back and remove the redundant muscle on it. Then open the spine from the central axis with rongeur, to ex- pose the spinal cord, on both sides of which is white nodule lies in intervertebral foraminas the DRG. We use tweezers damping inner nerve fiber,firstly cut the lateral one, later cut the inner one. The DRG is digested by collagenase combine Trypsin-EDTA. We seperate the DRG with different diamters of thick and thin pasteur glass tubes to get single neuron. Pri- mary culture these cells with the mixed TNB-100 basal Medium（ containing 2% Protein-Lipid-Complex, 10% FBS, 10% NGF, 1% antibiotic and 0.2 mM L-Glutamin）in petri dishes coated by 0. lmg/ml Poly-D-lysine hydrobromide plus 0.01 mg/ml lamina incubator for 48h. We detect the expression of Beta HI Tubulin, marker for neuron,by immunofluorescence staining, is labelled by Alexa flour 488 ： The second antibody were observe. The results under the inverted phase fluores- cence microscope. Results： The single layer adherent neurons, with different body shape were obtained. The branches from the body developed very well, connected with each other and gradually formed a network. The immunofluorescenee staining revealed that the cells bodies and branches can highly express Beta III Tubulin. Besides,the fluorescence staining ras very strong. Conclusion： Using this method can easily get the single layer adherent neurons coming from spinal cord dorsal root ganlions.

关 键 词：背根神经节(DRG) 原代培养 BetaⅢTubulin 大鼠 

分 类 号：R338[医药卫生—人体生理学]