17β-雌二醇对仔兔DRG神经元内Ca^(2+)浓度的影响

The Effect of 17β-estradiol on the Ca^(2+) Concentration in DRG Neurons of Newborn Rabbits

旨在研究雌激素能否影响初级感觉神经元调控活动以及Ca^(2+)在此影响机制中的作用。采用细胞免疫荧光技术观察仔兔背根神经节(DRG)神经元原代培养体系中雌激素受体(ER)的分布特点;采用激光共聚焦显微镜技术监测17β-E_2(1、10、100、1 000nmol·L^(-1))对体外培养72h仔兔DRG神经元细胞质内游离Ca^(2+)荧光强度的影响。结果表明,ERα、ERβ、GPR30在原代培养DRG神经元中分别有45.61%、32.39%、39.82%的阳性或强阳性神经元,且不同ER阳性或强阳性神经元中不同大小类型神经元所占比例不同;ERα、ERβ在DRG神经元细胞核为强阳性,细胞质为中等阳性,可见极少量弱阳性神经突起;GPR30在DRG神经元细胞核中为弱阳性或阴性,细胞质为中等阳性或强阳性,神经突起呈弱阳性。三种ER均在阳性反应神经元数量、大小类型、反应程度及分布部位上表现一定程度的"异质性";依据17β-E_2诱导神经元内Ca^(2+)荧光强度的变化特征,可将DRG培养体系中的神经元分为3种类型:兴奋型(19.28±0.70)%、抑制型(3.54±0.02)%和不敏感型(77.17±1.48)%;兴奋型神经元又可根据Ca^(2+)荧光变化强弱分为强兴奋型(0.195±0.118)和弱兴奋型(0.032±0.003)两种,随着17β-E_2浓度增大,强兴奋型神经元比例减少,但总兴奋型神经元比例各浓度组无显著性差异,体现出随17β-E_2浓度增大,对强兴奋型神经元[Ca^(2+)]_i增加幅度具有一定的抑制作用,但又非完全抑制。结果提示,(1)仔兔DRG神经元培养体系中神经元三种ER阳性反应的"异质性",表明雌激素可能对不同感觉神经元发挥影响效果不同;(2)Ca^(2+)通路是雌激素影响部分初级感觉神经元活动的作用机制之一。其次,雌激素可通过两种方式对DRG神经元胞内Ca^(2+)通路的效应发挥抑制性作用,一是可直接抑制少量神经元胞内Ca^(2+)信号通路效应;二是随浓度增大对强兴奋型神经元胞内[Ca^(2+)]_i增加所激活效应发挥一定的抑制作用。

The study was conducted to investigate whether estrogen can affect the regulation of primary sensory neurons,and to explore the role of Ca 2+in this mechanism.The distribution of estrogen receptor(ER)in primary culture dorsal root ganglion(DRG)from newborn rabbits was observed by Immunocytofluorescent(ICF),and the effects of 17β-E 2(1,10,100,1 000 nmol·L-1)on cytosolic free Ca 2+fluorescence in DRG neurons cultured for 72 h were monitored by Laser Scanning Confocal Microscopy(LSCM).It was found that,in primary cultured DRG neurons,ERα,ERβand GPR30 were 45.61%,32.39%,39.82% positive or strongly positive neurons respectively,and the neurons proportions of different sizes in different ER positive or strongly positive neurons were different.ERαand ERβwere strongly positive in DRG neuron nuclei,medium positive in cytoplasm,and weakly positive in a little neurite.GPR30 were weakly positive or negative in DRG neuron nuclei,medium positive or strongly positive in cytoplasm,and weakly positive in neurite.A certain degree of"heterogeneity"of three types of ERs(ERα,ERβ,and GPR30)was observed in the number,size type,degree of response and distribution in positive neurons.According to the variation of Ca^2+fluorescence intensity induced by 17β-E2,DRG neurons were divided into 3 types,excitatory(19.28±0.70)%,inhibitory(3.54±0.02)%and insensitive neurons(77.17±1.48)%,respectively.Excitatory neurons were divided into strongly excitatory neurons(0.195±0.118)and weakly excitatory neurons(0.032±0.003)depending on the intensity of Ca^2+fluorescence changes.Interestingly,with the increase of 17β-E2,the proportion of strongly excitatory neurons decreased,but no difference was detected in the proportion of total excitatory neurons at each group.It was indicated that with the increasing of 17β-E2,the increasement of[Ca^2+]i was inhibited in strongly excitatory neurons,but not completely inhibited.These results suggest that estrogen may have distinct effects on various sensory neurons;Ca^2+pathway is one of the mechanisms in the effects of estrogen on the activity of some primary sensory neurons,and estrogen inhibits the effect of Ca^2+in DRG neurons in two ways:a direct inhibition of the effect of Ca^2+signaling pathway in several neurons,and a certain inhibitory impact on the effect of Ca^2+increment in strongly excitatory neurons with the increasing estrogen.