利用原代培养神经元研究netrin-4的受体定位  

Location of the receptor for netrin-4 by primary culture of neurons of newborn rat

作　　者：余科科[1] 汪思应[1] 周仁平[2] 张成岗[3] 

机构地区：[1]安徽医科大学基础医学院,安徽合肥230032 [2]新泽西州立大学罗格斯院生物化学系,美国皮斯卡塔维nj08854 [3]军事医学科学院放射医学研究所,北京100850

出　　处：《基础医学与临床》2010年第7期726-730,共5页Basic and Clinical Medicine

基　　金：国家海外青年学者合作研究基金(30128010)

摘　　要：目的利用体外原代培养神经元,研究神经突起诱向因子netrin-4的受体,分析netrin-1已知受体DCC和UNC5H1作为netrin-4受体的可能性。方法改良Koh法培养出生12 h内Wistar乳鼠神经元,免疫细胞化学鉴定纯度;以碱性磷酸酶(AP)标记的netrin-4为配体,以神经元及转染DCC和UNC5H1的COS7细胞为对象,亲和细胞化学法检测netrin-4的受体;RT-PCR法分析神经元中netrin-4、DCC和UNC5H1的表达。结果成熟神经元胞核大,突起长,交织成网;以抗神经丝蛋白(NF)抗体行免疫细胞化学鉴定,纯度较高;转染DCC和UNC5H1的COS7能与AP4-netrin-4结合,定位在细胞膜上。结论建立起稳定的原代神经元培养法,并研究神经突起诱向因子受体的定位。DCC和UNC5H1可能是netrin-1与netrin-4的共受体。Objective To establish a primary culture model of neurons of newborn rat and to investigate whether the known netrin-1 receptors,DCC and UNC5H1,are the co-receptors for netrin-4.Methods We improved Koh method to cultivate the primary neurons of newborn rat.Evaluation of neurons was made by immunocytochemical techniques.Ligand-receptor binding assays and in situ staining with the alkaline phosphatase（AP4） tagged netrin-4 fusion protein were used.Reverse transcription polymerase chain reaction（RT-PCR） method was also used to observe the expression level of netrin-4,DCC,UNC5H1 mRNA in brain during development.Results Cultured neurons were stick to the wall of the cultivation plate.The neurons were satiated and with long axon.Immunocytochmical staining showed 90% neurofilament positive staining cell.In situ staining of AP4-netrin-4 protein bound to COS7 cells transiently transfected with eukaryotic expression coding DCC and UNC5H1 showed that the netrin-4 was bound to the transfected cells.Conclusion Primary culture of olfactory and cortical neurons of newborn rat is a reliable and stable method to obtain neurons.Primary culture of olfactory and cortical neurons of newborn rat is areliable and stable source for neurons.DCC and UNC5H1 are coreceptors for netrin-4.

关 键 词：神经元 受体 netrin-4 DCC UNC5H 

分 类 号：R341[医药卫生—基础医学]