不同糖浓度中大鼠DRG神经元相关蛋白表达及Ca2+浓度的测定

摘要

Objective To observe the effect of high glucose concentration on transient receptor potential vanilloid 4 (TRPV4),protein ki-nase C (PKCε )and intercellular Ca2+ concentration in DRG neurons of the rats and reveal the mechanism of painful diabetic neuropathy. Methods The DRG neurons were isolated from neonate rat. The neurons were divided into control(C),medium(M ) and high glucose(H) group which were primarily cultured in medium with different glucose concentration. After 48 h, the expression of TRPV4 and PKCε was de-tected by Western blotting. The Ca2+ concentration was detected by laser scanning confocal microscope. Results TRPV4 and PKCε in-creased in dose dependent manner in different glucose concentration. TRPV4 were 3.20±0.40 and 7.69±0.60 in M and H group,but C group was 0.33±0.05. PKCε showed 1.08±0.08 and 1.97±0.35 in M and H group,but C group was 0.88±0.04. 4α-PDD,a TRPV4 activator can raise intracellular calcium in dose-dependent manner,H group was significant (P< 0.05 vs control). Conclusion High glucose concentra-tion can active the expressions of TRPV4,PKCε in DRG neurons in rats. The responsibility of Ca2+ concentration of DRG neuron is accentu-ated in high glucose concentration. TRPV4 and PKCε both donate the augmented Ca2+ concentration in DRG neurons. High glucose levels in-crease the reactivity of the DRG neuron.%目的 观察高糖对背根神经节(DRG)中瞬时感受器电位香草酸受体4(TRPV4)和蛋白激酶Cε(PKCε)的表达以及细胞内Ca2+浓度的影响,探讨其在糖尿病大鼠神经病理性疼痛形成中的作用机制.方法 培养的新生大鼠DRG神经元分成正常对照组(C组)、中糖组(M组)和高糖组(H组).48 h后通过蛋白质免疫印迹法测定DRG神经元TRPV4和PKCε的表达水平,并通过共聚焦显微镜测定DRG神经元[Ca2+]i的水平.结果 TRPV4、PKCε蛋白的表达上调且呈浓度依赖性.C组、M组、H组TRPV4蛋白分别为0.33±0.05、3.20±0.40和7.69±0.60; PKCε蛋白分别为0.88±0.04、1.08±0.08和1.97±0.35.在TRPV4激动剂4α-PDD的作用下,与C组比较,H组[Ca2+]i显著升高(P＜0.05),且呈浓度依赖.结论 高糖可以上调DRG神经元中TRPV4、PKCε蛋白的表达,增加神经元内Ca2+浓度.TRPV4、PKCε对DRG神经元内Ca2+起到了协同调控的作用.