Wu Da-Yu, Zheng Jun-Qi, McDonald Marisa A, Chang Bieshia, Twiss Jeffery L
Departments of Cell and Neurobiology, Keck School of Medicine of the University of Southern California, Los Angeles, California 90033, USA.
Invest Ophthalmol Vis Sci. 2003 Jun;44(6):2783-90. doi: 10.1167/iovs.02-0715.
To establish an in vitro model of axonal regeneration from mammalian retinal ganglion cells and to evaluate the role of PKC isozymes in promoting such retinal axon regeneration.
Postnatal day-3 mice were subjected to optic nerve crush, and then retinal ganglion cells (RGCs) were used for culture 5 days later. RGCs were selected using anti-Thy 1.2-coated magnetic beads and plated onto a merosin substrate. Changes in axonal localization of PKC and axonal regeneration were examined in cultured RGCs by immunofluorescence. Changes in PKC isozyme mRNA levels were determined by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). The role of PKC in RGC neurite outgrowth was examined by treatment with activators or pharmacological inhibitors of PKC activity.
RGCs subjected to optic nerve crush injury demonstrated more rapid neurite outgrowth in vitro when compared with RGCs isolated from naïve retina. The neurites of these injury-conditioned RGCs showed both an increased rate of extension and enhanced PKC localization in culture. Injury-conditioned RGCs had elevated PKC isozyme mRNA levels, which probably contributed to the increased level of PKC protein in injury-conditioned RGC axons. Pharmacological activation of PKC enhanced neurite growth, whereas inhibition of PKC suppressed neurite growth in both the conditioned and naïve RGCs.
RGCs actively respond to axonal injury by regulating expression of genes that promote neurite outgrowth. PKC-alpha and -beta isozymes are among the growth-associated proteins that are upregulated after injury. Results of pharmacological manipulation of PKC activity support the argument that increased PKC levels enhance neurite regrowth after axonal injury.
建立哺乳动物视网膜神经节细胞轴突再生的体外模型，并评估蛋白激酶C（PKC）同工酶在促进视网膜轴突再生中的作用。
出生后第3天的小鼠接受视神经挤压，5天后使用视网膜神经节细胞（RGCs）进行培养。使用抗Thy 1.2包被的磁珠选择RGCs，并将其接种到巢蛋白底物上。通过免疫荧光检查培养的RGCs中PKC轴突定位的变化和轴突再生情况。通过半定量逆转录-聚合酶链反应（RT-PCR）测定PKC同工酶mRNA水平的变化。通过用PKC活性激活剂或药理学抑制剂处理来研究PKC在RGC神经突生长中的作用。
与从未损伤视网膜分离的RGCs相比，遭受视神经挤压损伤的RGCs在体外表现出更快的神经突生长。这些损伤条件下的RGCs的神经突在培养中显示出延伸速率增加和PKC定位增强。损伤条件下的RGCs的PKC同工酶mRNA水平升高，这可能导致损伤条件下的RGC轴突中PKC蛋白水平增加。PKC的药理学激活增强了神经突生长，而PKC的抑制则抑制了条件性和未损伤RGCs中的神经突生长。
RGCs通过调节促进神经突生长的基因表达来积极响应轴突损伤。PKC-α和-β同工酶是损伤后上调的生长相关蛋白之一。PKC活性的药理学操作结果支持了PKC水平升高增强轴突损伤后神经突再生的观点。
