腺病毒介导的BDNF基因在体外嗅鞘细胞中的表达及其生物学活性实验研究

BDNF Expression in the OECs Infected by Ad-BDNF in Vitro and Surviving the Primary Cultured Spinal Neurons

目的 :用重组腺病毒载体Ad BDNF感染体外培养的嗅鞘细胞 ,从基因转录和蛋白表达水平检测BDNF基因在体外OECs中的表达 ,对OECs表达的BDNF生物学活性进行观察。方法 :提取细胞总RNA进行RT PCR扩增BDNF基因 ,应用ELISA方法检测其培养上清中BDNF含量 ,并通过与原代脊髓神经元共培养观察BDNF活性。结果 :实验证实经BDNF重组腺病毒感染的OECs中有BDNF的转录 ;其培养上清应用ELISA方法分析 ,在感染后 2 4h即有BDNF的表达 ,并可以维持 3 5d的高峰表达。与BDNF重组腺病毒感染的OECs共培养的脊髓神经元可以在无血清条件下存活 3 5d。结论 :重组腺病毒BDNF基因可以在OECs中稳定、高效表达 ,其表达的BDNF具有促进脊髓神经元存活的生物活性。

Objective:To detect the expression of BDNF gene after the OECs infected by Ad-BDNF and to evaluate the activity of BDNF. Method:Three techniques immunohistochemistry, enzyme linked immunosorbent assay (ELISA) and reverse transcriptase-polymerase chain reaction (RT-PCR) were used to assess growth factor expression in cultured OECs.The primary spinal neurons were cultured with Ad-BDNF OECs without serum support. Result:Immunohistochemistry showed that OECs expressed brain derived neurotrophic factor (BDNF) from 24h of infection. ELISA confirmed the presence of BDNF in the liquid supernatant of OECs infected by Ad-BDNF and showed that BDNF was secreted. RT-PCR analysis demonstrated expression of mRNA for BDNF. The primary spinal neurons survived at least 5 DIV without serum support, while they died within 24～48 control group without Ad-BDNF OECs. Conclusion:The result of the experiments show that OECs express a number of growth factors BDNF at 20ng/ml.BDNF from the infected OECs can survive the primary cultured spinal neurons.