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

The Expression of Related Proteins and Calcium Level in DRG Neuron within Different Glucose Concentration

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。

Objective To observe the effect of high glucose concentration on transient receptor potential vanilloid 4 （TRPV4）, protein ki- nase C （PKCe）and intercellular Ca2＋ concentration in DRG neurons of the rots 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 PKCe was de- tected by Western blotting. The Ca2＋ concentration was detected by laser scanning confocal microscope. Results TRPV4 and PKCe in- creased in close 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. PKCE showed 1.08±0.08 and 1.97±0.35 in M and H grnup,bnt 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, PKCe in DRG neurons in mrs. The responsibility of Ca2＋ concentration of DRG neuron is accentu- ated in high glucose concentration. TRPV4 and PKCe both donate the augmented Ca2＋ concentration in DRG neurons. High glucose levels in- crease the reactivity of the DRG neuron.