嘌呤敏感途径影响视网膜节细胞轴突再生

A Purine-sensitive Signaling Pathway Controls Axon Regeneration in Goldfish Retinal Ganglion Cells

目的 探讨影响视网膜节细胞轴突再生的细胞内信号传递途径。方法 采用金鱼视网膜节细胞培养及Western blot观察几种神经营养因子对视网膜节细胞轴突再生的影响。结果 AF-1、AF-2、CNTF能刺激视网膜节细胞轴突再生及 GAP-43表达增强 ;6-硫鸟嘌呤 (6-TG)能抑制 AF-1、AF-2、CNTF对神经再生及 GAP-43表达的促进 ;肌苷不仅能刺激视网膜节细胞轴突再生及 GAP-43表达增强 ,而且能竞争性逆转 6-TG对 AF-1、AF-2、CNTF诱导神经再生的抑制。结论 上述几种神经营养因子通过一种嘌呤敏感途径的调节 ,能增强视网膜节细胞轴突再生及 GAP-43的表达。

Objective To investigate the signaling mechanism controlling axonal outgrowth in goldfish retinal ganglion cells (RGC).Methods RGC cultures:goldfish were dark adapted. Retinas were dissected, enzymatically digested and subjected to a series of trituration and sedimentation steps that resulted in a ganglion cell enriched suspension. Neurite outgrowth was assessed on the 6th day in 150 consecutive RGC per well using 400x magnification. Western blot:test procedure was carried out by RGC grown in the presence of various trophic factors or inhibitors for 5 days. Cells were collected, centrifuged, and their proteins were separated by SDS PAGE,and transferred to polyvinylidene difluoride membranes and probed with the primary antibody overnight at 4 °C, washed in TBS, and incubated for 1hr at RT using HRP conjugated secondary antibody followed by ECL reagent.Results In culture, RGC regenerated their axons in response to two molecules secreted by optic nerve glia, axogenesis factors (AF ) 1 and 2, along with ciliary neurotrophic factor (CNTF). Growth factor induced axogenesis was accompanied by molecular changes that characterize regeneration in vivo, e.g., increased expression of the membrane phosphoprotein GAP 43. The purine analog 6 thioguanine (6 TG) blocked outgrowth and inhibited the expression of GAP 43 induced by each of these factors. The purine nucleoside inosine had effects opposite to those of 6 TG. Inosine stimulated outgrowth and the expression of GAP 43 in RGC and competitively reversed the inhibitory effects of 6 TG. Conclusions The axonregeneration and the underlying molecular changes in RGC are mediated through a common, purine sensitive pathway, in the presence of above trophic factors.