Department of Neurobiology, Chongqing Key Laboratory of Neurobiology, Third Military Medical University, Chongqing 400038, People's Republic of China.
J Mol Neurosci. 2012 May;47(1):173-9. doi: 10.1007/s12031-012-9710-z. Epub 2012 Feb 9.
The primary sensory neurons with cell bodies in the dorsal root ganglion (DRG) have been extensively used as models in neurobiology and provide a useful model to study the mechanism of neural regeneration. Therefore, efficient and stable gene delivery to these postmitotic cells has significant therapeutic potential. Various studies involving the viral vector systems capable of neuronal transduction have been extensively evaluated in the cultured DRG neurons by adeno-associated virus. In the present study, we investigated the transduction performance of the lentiviral vector that mediates the catalytic subunit of protein kinase A (PKAc) and green fluorescent protein (GFP) expression in the DRG by sciatic nerve retrograde transport and tested whether PKAc expression in the DRG could inhibit the activation of RhoA after spinal cord injury. Five days after sciatic nerve remote delivery of lentiviral vector (LV)/PKAc-internal ribosome entry site (IRES)-GFP or LV/GFP, the L4-L6 DRGs were dissected for primary culture or immunostaining to observe the exogenous gene expression, or transecting the dorsal part of lumbar enlargement was performed, and 16 h later, the function of the exogenous gene was tested by RhoA pull-down analysis. The results showed that the lentiviral vector could mediate exogenous gene PKAc expression in the DRG and then inhibit spinal cord injury-induced RhoA activation by remote delivery of LV/PKAc-IRES-GFP through the sciatic nerve.
初级感觉神经元的细胞体位于背根神经节（DRG）中，已被广泛用作神经生物学的模型，并为研究神经再生机制提供了有用的模型。因此，将有效且稳定的基因递送到这些有丝分裂后细胞中具有重要的治疗潜力。通过腺相关病毒，在培养的 DRG 神经元中广泛评估了各种能够进行神经元转导的病毒载体系统的研究。在本研究中，我们通过坐骨神经逆行运输研究了介导蛋白激酶 A（PKAc）催化亚基和绿色荧光蛋白（GFP）表达的慢病毒载体在 DRG 中的转导性能，并测试了 DRG 中的 PKAc 表达是否能抑制脊髓损伤后 RhoA 的激活。在坐骨神经远程递送慢病毒载体（LV）/PKAc-内部核糖体进入位点（IRES）-GFP 或 LV/GFP 后 5 天，解剖 L4-L6 DRG 进行原代培养或免疫染色以观察外源性基因表达，或横断腰椎扩大部的背侧，16 小时后，通过 RhoA 下拉分析测试外源性基因的功能。结果表明，通过坐骨神经逆行递送 LV/PKAc-IRES-GFP，慢病毒载体可以介导 DRG 中外源基因 PKAc 的表达，然后抑制脊髓损伤诱导的 RhoA 激活。
