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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 lentiviral vector-mediated PKA catalytic subunit PKAc transfection to promote primary cultured adult rat dorsal root ganglion (DRG) neurite outgrowth growth and its mechanism. We co-transfected 293T cells with three plasmids to construct lentiviral vector LV / PKAc-IRES-GFP and control virus LV / GFP. The primary cultured DRG neurons were isolated and cultured by immunohistochemistry and Western blot cAMP / PKA signaling pathway downstream of the key transcription factor cAMP response element connexin (CREB) phosphorylation levels, image analysis of histochemical staining neurite length and percentage of neurons. The results showed that the lentivirus can induce foreign gene expression in primary cultured mammalian neurons. PC12 cells and DRG neurons transfected with LV / PKAc-IRES-GFP can express foreign genes and activate CREB, Overcome CNS myelin protein inhibition and promote the process of neurite outgrowth. The above experimental results show that rescuing cAMP / PKA levels in neurons of adult rats can effectively change the intrinsic growth ability of neurons, thereby changing their sensitivity to inhibition of the environment and further promoting the growth of neurites.