鸡胚脊髓背角感觉神经元诱向因子的初步分离及鉴定

为探讨脊髓背角内影响SG神经元存活和生长的因素,取 500只Hamburger 40期鸡胚脊髓背角组织匀浆,离心后提取上清液,用SDS-PAGE电泳初步分离脊髓背角内的蛋白质.以简易细胞钓蛋白带(PBFC)技术找寻有神经细胞贴附的蛋白带并求相对迁移率(Rf).将有神经细胞贴附的各Rf范围凝胶剪下并将胶内各蛋白带电转移至硝酸纤维膜上,用NGF、BDNF、NT-3、GDNF兔抗血清于硝酸纤维膜上分别进行Western杂交.PBF C结果显示: 在电泳胶Rf 0.10～0.19、0.30～0.35、0.50～0.55、0.60～0.67、0.90～0.94 处发现有多少不等的神经元附着.而Western杂交结果表明,在上述各Rf范围均有强弱不等的多条阳性杂交带.分别为NGF、BDNF、GDNF者;NGF、BDNF、NT-3者;NGF、BDNF、NT-3者 ;NGF、NT-3者、BDNF、NT-3者.提示:40期鸡胚脊髓背角提取液各Rf蛋白带的促神经元存活作用,至少涉及NGF、BDNF、NT-3、GDNF中的两种或三种.

Preliminary Isolation and Identification of Neurotrophic Factors Sensory Neurons in Spinal Dorsal Horn of Embryonic Chicken

Our previous studies have demonstrated that the extract from dorsal horn of embryonic chicken can promote apparently the neurite growth of spinal ganglion during development. In this study, we investigated progressively the neurotrophic factors for primary sensory neurons in spinal dorsal horn. The tissues of spinal dorsal horn from five hundred Hamburger Stage-40 embryonic chickens were taken. After homogenization and centrifugation, the supernatant was subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE ) for analyzing the protein in dorsal horn. The simple protein band fishing by cell (SPBFC) technique was employed in finding the protein bands with neurons attached, and then such protein bands, were taken and transferred to nitric cellulose paper for Western hybridization analysis by antibody for NGF, BDNF,NT-3 and GDNF respectively. The results of SPBFC showed that some neurons were attached to gel within the scope of mobility relative front (Rf) 0. 10-0. 19, 0. 30-0. 35, 0. 50-0. 55, 0. 60-0. 67 and 0. 90-0. 94 respectively, indicating there were some neurotrophic factors for maintaining neuron survival in the above Rf scope. Moreover, the result of Western hybridization showed that the positive hybridization bands in each scope were that of NGF, BDNF, GDNF;NGF,BDNF,NT-3;NGF, BDNF, NT-3; NGF, NT-3 and BDNF, NT-3 respectively;indicating that the neurotrophic effect of spinal dorsal horn tissue on primary sensory neurons involves NGF, BDNF,NT-3 and GDNF. These also suggest that the neuronal survival effect in each Rf scope involves at least two or more neurotrophic factors.