TRPV1和NR1之间的相互作用在利多卡因所致的背根神经元毒性中的研究

发布时间：2019-05-16 01:28

【摘要】：目的：检测利多卡因所致的背根神经元(dorsal root ganglion neurons, DRG neurons)毒性中TRPV1和NR1之间是否存在相互作用。方法：(1)用显微解剖方法获取足够数量的乳大鼠DRG,经胰酶消化处理后在体外进行纯化培养,采用免疫细胞化学染色法鉴定DRG神经元并计算其纯度；(2)用CCK-8法检测0、0.5、1、2、4和8mM利多卡因作用于DRG神经元30min时的生存率,并计算其半数致死浓度(Lethal Concentration 50, LC50);(3)用CCK-8法检测不同浓度的TRPV1激动剂capsacin、TRPV1特异性拮抗剂capsazepine、 NMDA受体激动剂NMDA、NMDA受体拮抗剂AP-5对LC50的利多卡因所致DRG神经元生存率的影响,取得其各自的最大效应浓度。(4)用western blot法检测并比较最大效应浓度的capsacin(100μM)、capsazepine(10μM)、 NMDA(200μM)和AP-5(100μM)对LC50的利多卡因所致DRG神经元TRPV1、NR1 (NMDA受体发挥功能的必须亚基)蛋白表达水平及其磷酸化水平的影响。结果：(1)经改进后的DRG神经元培养体系获得的DRG神经元在体外培养生长状态良好,纯度可达91%；(2)利多卡因对体外培养状态下的DRG神经元存在神经细胞毒性,浓度依赖性降低DRG神经元的生存率,其作用30min时的LCso为1.56mM;(3)与LC50利多卡因组比较：①一定浓度的capsacin可以降低LC50利多卡因所致的DRG神经元生存率,且100μM capsacin达最大效应浓度,将LC50利多卡因所致的DRG神经元生存率由50%降至40%(P0.05)；②一定浓度的capsazepine可以改善LC50利多卡因所致的DRG神经元生存率,且10μM capsazepine达最大效应浓度,将LC50利多卡因所致的DRG神经元生存率由50%上调至77%(P0.05); ③ NMDA可以降低LC50利多卡因所致的DRG神经元生存率,且200μM NMDA达最大效应浓度,使得LC50利多卡因所致的DRG神经元生存率由50%降至25%(P0.05)；④一定浓度的AP-5可以改善LC50利多卡因所致的DRG神经元生存率,且100μM的AP-5达最大效应浓度,将LC50利多卡因所致的DRG神经元生存率由50%上调至62%(P0.05)。(4)与LCso利多卡因组比较：①100μM capsacin加LCso利多卡因能显著增加利多卡因所致的DRG神经元的TRPV1和NR1蛋白的磷酸化水平(P0.05),而对DRG神经元TRPV1和NR1蛋白的表达未见明显影响；②10μM capsazepine加LCso利多卡因能显著降低利多卡因所致的DRG神经元的TRPV1和NR1蛋白的磷酸化水平(P0.05),而对DRG神经元TRPV1和NR1蛋白的表达未见明显影响；③200μM NMDA加LCso利多卡因能增加利多卡因所致的DRG神经元的NR1蛋白的磷酸化水平(P0.05),但对DRG神经元NR1和TRPV1蛋白的表达及TRPV1蛋白的磷酸化水平未见明显影响；④100μMAP-5加LCso利多卡因能够显著降低利多卡因所致的DRG神经元NR1蛋白的磷酸化水平(P0.05),而对DRG神经元NR1和TRPV1蛋白的表达及TRPV1蛋白的磷酸化水平未见明显影响。结论：(1)经改进后的DRG神经元体外培养方法可获得高质量、高纯度的DRG神经元,值得推广。(2)利多卡因对体外培养状态的DRG神经元具有神经毒性,其作用30min时的LCso为1.56mM。(3)TRPV1和NRl的磷酸化均参与了利多卡因所致的DRG神经元的毒性；在利多卡因所致的DRG神经元毒性中TRPV1的激活或抑制可明显影响NR1的磷酸化水平,而NRl的激活或抑制对TRPV1的磷酸化水平无明显调节作用。
[Abstract]:Objective: To investigate the interaction between TRPV1 and NR1 in the toxicity of dorsal root ganglion (DRG) induced by lidocaine. Methods: (1) A sufficient number of rat DRGs were obtained by microdissection. After the digestion, the DRG neurons were purified and cultured in vitro, and the DRG neurons were identified by immunocytochemical staining and their purity was calculated. (2) Using CCK-8 method to detect the survival rate of 0, 0.5,1,2,4 and 8 mM lidocaine on the DRG neurons for 30 min, and to calculate the half lethal concentration (Lethal concentration 50, LC50); and (3) to use the CCK-8 method to detect the different concentrations of the TRPV1 agonist capsacin, the TRPV1 specific antagonist capsazepine, the NMDA receptor agonist NMDA, The effect of the NMDA receptor antagonist AP-5 on the survival rate of the DRG neurons induced by Lidocaine in the LC50 results in their respective maximum effect concentrations. (4) The effect of capsuacin (100. mu.M), capsanthin (10. mu.M), NMDA (200. mu.M) and AP-5 (100. mu.M) on the expression level and phosphorylation of the DRG neurons, TRPV1, NR1 (the essential subunit of the function of the NMDA receptor), was detected and compared with western blot. Results: (1) The DRG neurons obtained by the improved DRG neuron culture system had good growth state in vitro, and the purity can reach 91%; (2) Lidocaine has the toxicity of the nerve cells in the DRG neurons in the in vitro culture state, and the concentration dependence reduces the survival rate of the DRG neurons. The LCso was 1.56 mM when it was used for 30 min, and (3) compared with the L50 lidocaine group: a certain concentration of capsuacin could reduce the survival rate of the DRG neurons due to the LC50 lidocaine, and the survival rate of the DRG neurons due to the LC50 lidocaine decreased from 50% to 40% (P0.05). At a certain concentration of capsuzerine, the survival rate of the DRG neurons due to the LC50 lidocaine can be improved, and the survival rate of the DRG neurons due to the LC50 lidocaine is up to 77% by 50% (P0.05), and the survival rate of the DRG neurons due to the LC50 lidocaine can be reduced by the administration of NMDA. and the survival rate of the DRG neurons caused by the LC50 lidocaine is reduced from 50% to 25% (P0.05), and the AP-5 with a certain concentration can improve the survival rate of the DRG neurons caused by the LC50 lidocaine, and the AP-5 of 100. m The survival rate of DRG neurons due to L50 lidocaine was increased from 50% to 62% (P0.05). (4) Compared with LCso Lidocaine group, the level of the phosphorylation of TRPV1 and NR1 in the DRG neurons due to lidocaine can be significantly increased by 100. m u.M capsuacin plus LCso Lidocaine (P0.05), and the expression of TRPV1 and NR1 in the DRG neurons was not significantly affected. 10. m u.M capsuazepine and LCso lidocaine could significantly lower the level of the phosphorylation of TRPV1 and NR1 in the DRG neurons induced by lidocaine (P0.05), while the expression of TRPV1 and NR1 in the DRG neurons was not significantly affected. The expression of NR1 and TRPV1 in DRG neurons and the phosphorylation of TRPV1 protein were not significantly affected by the addition of 200. m u.M of NMDA and LCso lidocaine to the level of phosphorylation of the NR1 protein in the DRG neurons by lidocaine (P0.05). The expression of NR1 and TRPV1 in the DRG neurons and the phosphorylation of the TRPV1 protein were not significantly affected by the addition of 100 & mu; MAP-5 and LCso Lidocaine. Conclusion: (1) The improved DRG neurons can obtain high-quality, high-purity DRG neurons. (2) Lidocaine had neurotoxicity on the DRG neurons in vitro, and LCso was 1.56 mM at 30 min. (3) The phosphorylation of TRPV1 and NRl was involved in the toxicity of the DRG neurons induced by lidocaine; activation or inhibition of TRPV1 in the toxicity of the DRG neurons caused by lidocaine can significantly influence the phosphorylation level of NR1, while the activation or inhibition of NRl does not have a significant effect on the phosphorylation level of TRPV1.
【学位授予单位】：东南大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R614

【参考文献】