N-花生四烯基乙醇胺对背根神经节轴突再生的影响

Effect of N-arachidonylethanolamine on axon regeneration of the dorsal root ganglion

背景:神经轴突再生主要受其自身再生能力和抑制性外环境的影响,N-花生四烯基乙醇胺在秀丽线虫中已被证实可以调控神经轴突再生,而在哺乳动物中的作用仍是未知。目的:探究N-花生四烯基乙醇胺对小鼠背根神经节神经元轴突再生的作用。方法:取6-8周龄ICR小鼠的L4-L5处背根神经节组织,经过胶原酶和胰酶消化后,分成脂肪酸酰胺水解酶选择性抑制剂URB597干预组、N-花生四烯基乙醇胺干预组、二甲亚砜对照组、电转绿色荧光蛋白与特异性脂肪酸酰胺水解酶siRNA混合物组。细胞经过3 d的体外培养后,通过神经元特异性抗体免疫标记轴突,测量轴突长度、初级分支和次级分支数量并对其进行统计,判断N-花生四烯基乙醇胺对背根神经节神经元轴突再生的调控作用。结果与结论:①抑制脂肪酸酰胺水解酶活性后,背根神经节细胞轴突再生没有明显的变化并且URB597对背根神经节神经元不具有细胞毒性作用(P>0.05);②敲减脂肪酸酰胺水解酶后,背根神经节细胞轴突再生没有明显的变化(P>0.05);③外源性N-花生四烯基乙醇胺对背根神经节细胞轴突再生没有明显的调控作用,并且N-花生四烯基乙醇胺,对背根神经节神经元细胞没有细胞毒性作用(P>0.05);④无论是抑制脂肪酸酰胺水解酶活性还是加入外源性N-花生四烯基乙醇胺,对背根神经节神经元轴突分支均没有影响(P>0.05)。

BACKGROUND:Nerve axon regeneration is mainly affected by its own regenerative ability and inhibitory external environment.N-arachidonylethanolamine has been proven to regulate axon regeneration in C.elegans,but its role in mammals is still unknown.OBJECTIVE:To explore the effect of N-arachidonylethanolamine on axon regeneration of mouse dorsal root ganglion neurons.METHODS:Dorsal root ganglion tissues were taken from L_(4)-L_(5) of ICR mice aged 6-8 weeks,digested with collagenase and trypsin,and divided into URB597 group,N-arachidonylethanolamine group,methyl-sulfoxide control group,and electroporated green fluorescent protein and specific siRNA mixture group.After 3 days of in vitro culture,the axons were labeled by neuron-specific immunoassay.Axon length,and the number of primary and secondary branches were measured,and the statistics was performed to determine the regulatory effect of N-arachidonylethanolamine on the regeneration of dorsal root ganglion neuron axon.RESULTS AND CONCLUSION:(1)After inhibiting fatty acid amide hydrolase activity,there was no obvious change in dorsal root ganglion cell axon regeneration and URB597 had no cytotoxic effect on dorsal root ganglion neuron cells(P>0.05).(2)After fatty acid amide hydrolase knockdown,dorsal root ganglion cell axon regeneration had no obvious change(P>0.05).(3)Exogenous N-arachidonylethanolamine had no obvious regulatory effect on dorsal root ganglion cell axon regeneration and N-arachidonylethanolamine had no cytotoxic effect on dorsal root ganglion neuron cells(P>0.05).(4)Inhibiting fatty acid amide hydrolase activity or adding exogenous N-arachidonylethanolamine has no effect on dorsal root ganglion neuron axon branches(P>0.05).