TRPV1和NR1之間的相互作用在利多卡因所致的背根神經(jīng)元毒性中的研究
發(fā)布時間：2019-05-16 01:28
【摘要】：目的：檢測利多卡因所致的背根神經(jīng)元(dorsal root ganglion neurons, DRG neurons)毒性中TRPV1和NR1之間是否存在相互作用。方法：(1)用顯微解剖方法獲取足夠數(shù)量的乳大鼠DRG,經(jīng)胰酶消化處理后在體外進行純化培養(yǎng),采用免疫細胞化學染色法鑒定DRG神經(jīng)元并計算其純度；(2)用CCK-8法檢測0、0.5、1、2、4和8mM利多卡因作用于DRG神經(jīng)元30min時的生存率,并計算其半數(shù)致死濃度(Lethal Concentration 50, LC50);(3)用CCK-8法檢測不同濃度的TRPV1激動劑capsacin、TRPV1特異性拮抗劑capsazepine、 NMDA受體激動劑NMDA、NMDA受體拮抗劑AP-5對LC50的利多卡因所致DRG神經(jīng)元生存率的影響,取得其各自的最大效應濃度。(4)用western blot法檢測并比較最大效應濃度的capsacin(100μM)、capsazepine(10μM)、 NMDA(200μM)和AP-5(100μM)對LC50的利多卡因所致DRG神經(jīng)元TRPV1、NR1 (NMDA受體發(fā)揮功能的必須亞基)蛋白表達水平及其磷酸化水平的影響。結果：(1)經(jīng)改進后的DRG神經(jīng)元培養(yǎng)體系獲得的DRG神經(jīng)元在體外培養(yǎng)生長狀態(tài)良好,純度可達91%；(2)利多卡因對體外培養(yǎng)狀態(tài)下的DRG神經(jīng)元存在神經(jīng)細胞毒性,濃度依賴性降低DRG神經(jīng)元的生存率,其作用30min時的LCso為1.56mM;(3)與LC50利多卡因組比較：①一定濃度的capsacin可以降低LC50利多卡因所致的DRG神經(jīng)元生存率,且100μM capsacin達最大效應濃度,將LC50利多卡因所致的DRG神經(jīng)元生存率由50%降至40%(P0.05)；②一定濃度的capsazepine可以改善LC50利多卡因所致的DRG神經(jīng)元生存率,且10μM capsazepine達最大效應濃度,將LC50利多卡因所致的DRG神經(jīng)元生存率由50%上調至77%(P0.05); ③ NMDA可以降低LC50利多卡因所致的DRG神經(jīng)元生存率,且200μM NMDA達最大效應濃度,使得LC50利多卡因所致的DRG神經(jīng)元生存率由50%降至25%(P0.05)；④一定濃度的AP-5可以改善LC50利多卡因所致的DRG神經(jīng)元生存率,且100μM的AP-5達最大效應濃度,將LC50利多卡因所致的DRG神經(jīng)元生存率由50%上調至62%(P0.05)。(4)與LCso利多卡因組比較：①100μM capsacin加LCso利多卡因能顯著增加利多卡因所致的DRG神經(jīng)元的TRPV1和NR1蛋白的磷酸化水平(P0.05),而對DRG神經(jīng)元TRPV1和NR1蛋白的表達未見明顯影響；②10μM capsazepine加LCso利多卡因能顯著降低利多卡因所致的DRG神經(jīng)元的TRPV1和NR1蛋白的磷酸化水平(P0.05),而對DRG神經(jīng)元TRPV1和NR1蛋白的表達未見明顯影響；③200μM NMDA加LCso利多卡因能增加利多卡因所致的DRG神經(jīng)元的NR1蛋白的磷酸化水平(P0.05),但對DRG神經(jīng)元NR1和TRPV1蛋白的表達及TRPV1蛋白的磷酸化水平未見明顯影響；④100μMAP-5加LCso利多卡因能夠顯著降低利多卡因所致的DRG神經(jīng)元NR1蛋白的磷酸化水平(P0.05),而對DRG神經(jīng)元NR1和TRPV1蛋白的表達及TRPV1蛋白的磷酸化水平未見明顯影響。結論：(1)經(jīng)改進后的DRG神經(jīng)元體外培養(yǎng)方法可獲得高質量、高純度的DRG神經(jīng)元,值得推廣。(2)利多卡因對體外培養(yǎng)狀態(tài)的DRG神經(jīng)元具有神經(jīng)毒性,其作用30min時的LCso為1.56mM。(3)TRPV1和NRl的磷酸化均參與了利多卡因所致的DRG神經(jīng)元的毒性；在利多卡因所致的DRG神經(jīng)元毒性中TRPV1的激活或抑制可明顯影響NR1的磷酸化水平,而NRl的激活或抑制對TRPV1的磷酸化水平無明顯調節(jié)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.
【學位授予單位】：東南大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R614
本文編號：2477912
[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.
【學位授予單位】：東南大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R614
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