神经再生素促大鼠海马神经元生长的研究

目的 研究牛膝提取物神经再生素(NRF)对体外培养的大鼠海马神经元生长的促进作用及其对生长相关蛋白-43(GAP-43)表达的影响.方法 以体外原代培养的胎鼠海马神经元为研究模型,通过相差显微镜采用Scion软件测量不同浓度NRF(0.25mg/L、0.5mg/L、1mg/L)、不同作用时间(6h、12h、24h)对海马神经元神经突起生长的影响;通过实时荧光定量PER观察NRF不同浓度(0.25mg/L、0.5mg/L、1.0mg/L)及不同作用时间(6h、12h、24h)对海马神经元GAP-43基因表达的影响;采用免疫荧光细胞化学法和Western blotting,观察不同浓度NRF(0.25mg/L、0.5mg/L、1.0mg/L)加药24h后,对海马神经元GAP-43表达的影响.结果 NRF可有效地促进海马神经元的生长,加药后24h作用浓度为1mg/L时,作用最强;实时荧光定量RT-PCR、免疫荧光细胞化学法和Western blotting的结果提示,NRF能增加体外培养的海马神经元GAP-43的表达,浓度为1.0mg/L时,作用最佳.结论 NRF能促进体外培养的海马神经元神经突起的生长和GAP-43基因的表达,表明NRF对海马神经元具有神经营养作用.

EFFECTS OF NERVE REGENERATION FACTOR ON THE NEURITE GROWTH OF RAT HIPPOCAMPAL NEURONS

Objective To determine the effects of nerve regeneration factor(NRF) on neurite growth in cultured hippocampal neurons of rats. Methods After different periods(6h,12h,24h) of incubation by different concentrations(0.25mg/L,0.5mg/L,1.0mg/L) of NRF,the hippocampal neurons were observed and photographed by phase contrast microscopy,and the neurite length was analyzed using the Scion software.Real-time fluorescence quantitative RT-PCR was performed to examine the mRNA levels of growth associated protein 43(GAP-43) after different incubation periods with different concentrations of NRF.Fluorescent immunocytochemistry and Western blotting were performed to further examine the protein levels of GAP-43 in cultured hippocampal neurons after 24h incubation with different concentrations of NRF. Results NRF promoted the neurite growth of cultured hippocampal neurons with the most obvious mophological changes appearing upon treatment with NRF at 1mg/L for 24 hours.Real-time fluorescence quantitative RT-PCR showed that the mRNA level of GAP-43 in cultured hippocampal neurons was up-regulated by NRF in a dose and time-dependent manner, with the best concentration of 1mg/L.Meanwhile,fluorescent immunocytochemistry and Western blotting showed that the protein level of GAP-43 was up-regulated by NRF with the best concentration of 1mg/L.Conclusion NRF could promote the neurite growth and modulate the expression of GAP-43 in cultured hippocampal neurons,suggesting neurotrophic effects of NRF.