PKAc促进成年大鼠背根神经节神经元突起生长及其机制的研究

Study on the promotion of adult rat DRGNs neurite outgrowth by protein kinase A catalytic subunit and its mechanism

观察慢病毒载体介导PKA催化亚单位PKAc转染促进成年大鼠原代培养的背根神经节(DRG)神经元突起生长及其机制的研究。我们运用三质粒系统共转染293T细胞合成慢病毒载体LV/PKAc-IRES-GFP和对照病毒LV/GFP,原代分离培养成年大鼠DRG神经元,应用免疫组织化学染色和Western blot等方法检测cAMP/PKA信号通路下游关键转录因子cAMP反应元件连接蛋白(CREB)磷酸化水平,图像分析经组织化学染色后的神经元突起长度和有突起神经元的百分比。结果观察到,慢病毒能介导外源基因在哺乳动物原代培养的神经元中表达,转染LV/PKAc-IRES-GFP的PC12细胞和DRG神经元均能表达外源基因并激活CREB,克服CNS髓鞘蛋白的抑制,促进突起生长。以上实验结果表明拯救成年大鼠神经元cAMP/PKA水平可有效改变神经元内在生长能力,从而改变它们对抑制环境的敏感性,进而促进突起生长。

To observe the neurite outgrowth of adult rat dorsal root ganglion neurons （DRGNs） promoted by Lentiviral vector （LV） mediated protein kinase A （PKA） catalytic subunit transfection and to study its mechanism. Lentiviral stock was produced using threeplasmid system by transfecting HEK 293T cells. Adult rat DRG neurons were primary cultured, immunohistostaining and Western blot were used to test the phosphorylated level of a key transcript factor of cAMP/PKA signal pathway downstream, cAMP response element binding protein （CREB）. Image analysis was used to analyze the neurite length and percentage of neurons bearing neurites after immunohistostaining. The results showed that Lentiviral vector could mediate the exogeneous gene expression in mammalian primary culture neuron. After transfecting LV/PKAc-IRES-GFP, both the PC12 cell and DRGNs expressed exogeneous gene which activate CREB, The neurons transfecting with LV/PKAc-IRES-GFP could overcome the inhibition of CNS myelin and grow on myelin substrate. So the present results demonstrate that rescuing the cAMP/PKA level of adult neurons could change effectively its intrinsic growth capacity which insensitive to the inhibitory signal in the environment and promote neurite outgrowth.