牛膝多肽促大鼠海马神经元突起的生长

Polypeptides of Achyranthes bidentata Blume promotes the neurite growth of rat hippocampal neurons

目的研究牛膝多肽(ABPP)对体外培养的大鼠海马神经元突起生长的促进作用及其对生长相关蛋-白43(GAP-43)和神经丝蛋白(NF-H)mRNA和蛋白表达的影响。方法以体外原代培养的胎鼠海马神经元为研究模型,通过四甲基偶氮唑盐(MTT)检测ABPP对海马神经元细胞活性的影响,通过免疫荧光细胞化学法,采用Image-Pro Express软件测量不同浓度ABPP(0.1mg/L、0.5mg/L、1.0mg/L)加药24h,对海马神经元神经突起生长的影响;通过实时荧光定量PCR法定量分析不同浓度ABPP(0.1mg/L、0.5mg/L、1.0mg/L)加药6h,对海马神经元GAP-43和NF-H基因表达的影响;采用免疫印迹法观察不同浓度ABPP(0.1mg/L、0.5mg/L、1.0mg/L)加药24h后,对海马神经元GAP-43和NF-H蛋白表达的影响。同时应用胞外信号调节激酶(ERK)特异性拮抗剂PD98059与ABPP共培养海马神经元,分析ABPP对海马神经元的作用与ERK通路的关系。结果ABPP可有效地促进海马神经元突起的生长,加药后24h浓度为1.0mg/L,作用最强;实时荧光定量RT-PCR、免疫荧光细胞化学法和免疫印迹检测的结果表明,ABPP能增加体外培养的海马神经元GAP-43和NF-H的mRNA和蛋白表达,PD98059可抑制该过程。结论ABPP能促进体外培养的海马神经元神经突起的生长,其作用与上调GAP-43和NF-H基因的表达相关,并可能与ERK通路有关。

Objective To determine the effects of polypeptides of Achyranthes bidentata Blume （ABPP） on neurite growth in cultured hippocampal neurons of rats. Methods The cell activity of rat hippocampal, neurons with ABPP was tested with MTY. After 24 hours of incubation with different concentrations （0.1 mg/L, 0.5 mg/L, 1.0 mg/L） of ABPP, the hippocampal neurons were photographed by TCS SP2 confocal microscope with fluorescent immunocytochemistry, and the neurite length was analyzed using the Image-Pro Express software. Real-time fluorescence quantitative RT-PCR was performed to examine the mRNA levels of GAP-43 and NF-H after 6 hour with different concentrations of ABPP. Western blotting was performed to further examine the protein levels of GAP-43 and NF-H in cultured hippocampal neurons after 24 hours incubation with different concentrations of ABPP. Results ABPP promoted the neurite growth of cultured hippocampal neurons. Real-time fluorescence quantitative RT-PCR showed that the mRNA level of GAP-43 and NF-H in cultured hippocampal neurons was up-regulated by ABPP in a dose dependent manner. Meanwhile, fluorescent immunocytochemistry and Western blotting showed that the protein level of GAP-43 and NF-H were up-regulated by ABPP. Conclusion ABPP could promote the neurite growth and modulate the expression of GAP-43 and NF-H in cultured hippocampal neurons, which maybe have something to do with ERK pathway.