中脑导水管周围灰质D-丝氨酸在大鼠电针耐受中的作用

发布时间：2018-06-07 02:00

本文选题：中脑导水管周围灰质腹外侧区 + 电针耐受　；参考：《遵义医学院》2017年硕士论文

【摘要】：NMDA受体(N-甲基-D-天冬氨酸受体,N-methyl-D-aspartate receptor)是离子型谷氨酸受体的一个亚型,在中枢神经系统发育及多种神经系统疾病中发挥重要的生理及病理功能。NMDA受体NR1亚基上存在“甘氨酸位点(glycine site)”,该位点被甘氨酸结合后,谷氨酸(glutamic acid)才能有效开放NMDA受体偶联的离子通道。多年研究显示,D-丝氨酸(D-serine,D-Ser)才是NMDA受体NR1亚基“甘氨酸位点”的内源性配体,D-Ser通过调节NMDA受体的激活参与机体多种生理及病理过程,其中包括痛觉信息的调制过程。针对中枢神经系统D-Ser功能的研究近年已成为神经科学领域研究的热点之一。电针(electroacupuncture,EA)是将传统针灸与电疗相结合,进而产生临床镇痛作用,但长时间或反复多次给予电针刺激,电针的镇痛效应会逐渐减低以致消失,表现为电针耐受(electroacupuncture tolerance,EA tolerance),限制其在临床中的运用。中脑导水管周围灰质(periaqueductal gray,PAG)是机体内源性镇痛系统的关键部位,特别是其腹外侧区(ventrolateral periaqueductal gray,vl PAG),在疼痛调制和吗啡耐受的形成、维持机制中均有重要作用。大量研究显示,吗啡耐受和电针耐受之间存在交叉耐受,但在vl PAG中D-Ser是否可以通过调节NMDA受体功能参与电针耐受的形成和维持机制,这一点未见文献报道。本研究旨在明确在vl PAG中D-Ser是否介入大鼠电针耐受的形成和维持机制。目的:观察大鼠中脑导水管周围灰质腹外侧区(vl PAG)中D-Ser在大鼠电针耐受形成和维持中的作用并探讨其初步的中枢神经化学机制。方法:SD大鼠外周穴位给予多次电针刺激后建立大鼠电针耐受模型;(1)观察电针耐受大鼠机械痛阈(MWT)、热痛阈(TWL)变化百分数的改变;(2)ELISA法检测大鼠在电针耐受形成前后vl PAG中D-Ser水平变化;(3)免疫组化、免疫印迹法检测大鼠电针形成前后vl PAG中SR的表达部位及蛋白表达量的变化情况;(4)免疫组化法检测大鼠电针耐受形成前后vl PAG中星形胶质细胞活化的情况;(5)电针耐受大鼠vl PAG微量注射不同剂量的星形胶质细胞抑制剂-氟代柠檬酸(Fc)后再行电针刺激,观察大鼠MWT、TWL变化百分数变化并检测D-Ser水平变化情况。结果:在各观察时间点,假电针组大鼠MWT、TWL变化百分数与正常组大鼠相比较,差异无统计学意义(P0.05);电针组大鼠MWT、TWL变化百分数与正常组比较显著升高(P0.001),但随着电针刺激次数的增多,MWT、TWL变化百分数逐渐下降,6次电针后MWT、TWL趋近于正常组痛阈变化百分数值,P0.05,提示:大鼠电针耐受形成。同一时间点,电针耐受大鼠vl PAG中D-Ser水平升高、SR阳性细胞数和SR蛋白含量表达上调(P0.001),且vl PAG中星形胶质细胞活化表达量增多,与正常组、假电针组比较,差异具有统计学意义(P0.001);电针耐受大鼠vl PAG中微量注射不同剂量的Fc后再给予电针刺激,星型胶质细胞活化受抑制,vl PAG中D-Ser水平下降,大鼠MWT、TWL变化百分数升高,差异具有统计学意义(P0.001),提示大鼠电针耐受缓解。结论:vl PAG中星形胶质细胞活化引起D-Ser释放增加参与大鼠电针耐受机制。
[Abstract]:The NMDA receptor (N- methyl -D- aspartic acid receptor, N-methyl-D-aspartate receptor) is a subtype of the ionotropic glutamate receptor, which plays an important physiological and pathological function in the central nervous system and a variety of nervous system diseases. The "glycine acid site (glycine site)" exists on the NR1 subunit of the.NMDA receptor NR1, which is binding to glycine Then, glutamic acid (glutamic acid) can effectively open the ion channel coupled with NMDA receptor. Years of studies have shown that D- serine (D-serine, D-Ser) is the endogenous ligand of the NMDA receptor NR1 subunit "glycine site". D-Ser participates in various physiological and pathological processes by regulating activation of NMDA receptors, including modulation of pain information. The study of the D-Ser function of the central nervous system (CNS) has become one of the hot spots in the field of neuroscience in recent years. Electroacupuncture (EA) combines traditional acupuncture and electrotherapy with electrotherapy to produce clinical analgesic effect. However, electroacupuncture has been given for a long time or repeatedly, and the analgesic effect of electroacupuncture will gradually decrease and disappear. Electroacupuncture tolerance (EA tolerance), which restricts its application in clinical practice. The periaqueductal gray (PAG) around the mesencephalic aqueduct is the key part of the body's endogenous analgesic system, especially in the ventral lateral region (ventrolateral periaqueductal gray, VL PAG), in the form of pain modulation and morphine tolerance There is an important role in the maintenance mechanism. A large number of studies have shown that there is a cross tolerance between morphine tolerance and electroacupuncture tolerance, but whether D-Ser can be involved in the formation and maintenance of electroacupuncture tolerance by regulating NMDA receptor function in VL PAG is not reported in the literature. This study aims to determine whether D-Ser is involved in rat electricity in VL PAG. The formation and maintenance mechanism of needle tolerance. Objective: To observe the role of D-Ser in the formation and maintenance of electroacupuncture tolerance in rat mesencephalic periaqueductal gray matter (VL PAG) and to explore its primary central neurochemical mechanism. Methods: the rat electroacupuncture tolerance model was established after multiple electroacupuncture stimulation in the peripheral acupoints of SD rats; (1) observation of electricity. The needle tolerance of mechanical pain threshold (MWT) and thermal pain threshold (TWL) change percentage of rats; (2) ELISA method was used to detect the change of D-Ser level in VL PAG in rats before and after the formation of electroacupuncture tolerance; (3) immunoblotting and immunoblotting were used to detect the expression of SR in VL PAG before and after the formation of electroacupuncture in rats; (4) immuno histochemistry was used to detect large amounts. The activation of astrocytes in VL PAG before and after formation of rat Electroacupuncture; (5) electroacupuncture tolerance rats VL PAG microinjection of different doses of astrocyte inhibitor - fluorocitric acid (Fc) after electroacupuncture stimulation, observe the change of MWT and TWL changes in rats and examine the change of D-Ser level. Results: at each observation time point, false There was no significant difference in the change percentage of MWT and TWL in the electroacupuncture group with the normal group (P0.05). The percentage of MWT in the electroacupuncture group was significantly higher than that in the normal group (P0.001), but with the increase of the number of stimulation of the electroacupuncture, the percentage of MWT and TWL decreased gradually, and MWT after 6 times electroacupuncture, TWL reached a hundred of the normal group pain threshold. P0.05, suggested that the electroacupuncture tolerance of rats was formed. At the same time point, the D-Ser level in the electroacupuncture tolerance rats increased in the VL PAG, the number of SR positive cells and the expression of SR protein up up (P0.001), and the activation expression of astrocytes in VL PAG increased, and the difference was statistically significant compared with the normal group and the false electroacupuncture group (P0.001); Electroacupuncture tolerance was large. Rat VL PAG was injected with different doses of Fc and stimulated by electroacupuncture, the activation of astrocytes was inhibited, D-Ser level in VL PAG decreased, the percentage of MWT and TWL in rats increased, the difference was statistically significant (P0.001), suggesting that the rat electroacupuncture tolerance remission. Conclusion: astrocyte activation in VL PAG leads to increased participation of astrocyte activation in D-Ser release. The mechanism of electroacupuncture tolerance in rats.
【学位授予单位】：遵义医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R245

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