地佐辛对糖尿病神经病理性疼痛大鼠脊髓背角NMDA-2B受体表达的影响

发布时间：2019-06-19 22:27

【摘要】：目的：糖尿病周围神经病变（diabetic peripheral neuropathy, DPN）疼痛属于神经病理性疼痛（neuropathic pain, NP)的一种类型，DPN是糖尿病常见的并发症之一，在糖尿病患者中的发病率约为7.5%-24%。DPN的发病机制是非常复杂的，中枢敏化在DPN的产生和维持中起到了重要的作用，而兴奋性神经递质谷氨酸及其受体NMDA参与了中枢敏化的发生。NMDA-2B作为NMDA受体的一个亚基，对伤害性信号的传递尤其重要，因此，近年来的对NMDA受体的研究重点放在NMDA-2B亚基上。地佐辛作为新型人工合成强效的混合型阿片受体激动-拮抗剂，其特点是镇痛作用强而不良反应低，已在临床广泛应用。研究发现弱阿片受体激动剂曲马多对DPN疼痛治疗是有效的，而地佐辛对DPN的疗效目前并不十分清楚，本实验通过腹腔注射链尿佐菌素（STZ），制备糖尿病神经病理性疼痛大鼠模型，然后给予地佐辛进行干预治疗。通过观察正常组（Normal组）、糖尿病神经病理性疼痛组（Control组）以及糖尿病神经病理性疼痛模型制备成功后地佐辛干预组（Dezocine组）三组大鼠的体重、空腹血糖和50%缩足反应阈值（PWT）的变化，采用免疫组织化学染色技术以及RT-PCR技术，比较正常大鼠、糖尿病神经病理性疼痛大鼠以及地佐辛干预的糖尿病神经病理性疼痛大鼠的脊髓背角NMDA-2B受体的表达变化，从而探讨地佐辛对糖尿病神经病理性疼痛大鼠疼痛的影响及其可能的作用机制。方法：健康成年雄性SD大鼠80只，体重130g～150g，由河北医科大学动物实验中心提供。模型的制备：随机选择60只大鼠腹腔注射STZ（50mg/kg），注射STZ一周以后，有45只大鼠的空腹血糖16.7mol/L（成功率为75%），达到合格的标准。注射STZ两周后，用von Frey Hairs测定实验大鼠的PWT，PWT小于4g为合格的神经病理性疼痛大鼠模型。剩余的20只大鼠作为正常对照大鼠组，用同等体积的生理盐水代替STZ腹腔注射。取糖尿病神经病理性疼痛大鼠36只，随机分为2组，每组18只：一组为地佐辛组（Dezocine组），于注射STZ后第2周开始大鼠大腿外侧肌肉注射地佐辛2.52mg/kg，每日一次，连续肌肉注射1周；一组为糖尿病神经病理性疼痛大鼠组（Control组），用同等体积的生理盐水代替地佐辛，方法同前；取正常大鼠18组，作为正常组（Normal组），，用同等体积的生理盐水代替地佐辛，方法同前。分别于注射STZ前（T0）、注射后第2周（T1）、第3周（T2）、第5周（T3）、第7周（T4）5个时间点测量各组大鼠的体重、空腹血糖、PWT，于T2、T3、T4时间点三组分别随机抽取6只大鼠取材。其中3只大鼠用于免疫组织化学染色技术，麻醉后用4%的多聚甲醛液灌流固定后，取大鼠胸腰段膨大处的脊髓标本制作成蜡块，进行免疫组织化学染色，测定大鼠脊髓背角NMDA-2B受体的免疫阳性神经细胞数；另外3只大鼠麻醉后快速在提取大鼠胸腰段膨大处脊髓组织，利用PT-PCR技术半定量测定NMDA-2B受体的mRNA含量。采用SPSS17.0统计学软件进行分析，计量资料均以平均数±标准差(x _±s)表示，组内比较采用单因素方差分析，组间比较采用t检验，P0.05表示差异有统计学意义。结果： 1三组大鼠在体重、空腹血糖、50%缩足反应阈值方面的变化比较：注射STZ前，三组大鼠体重、空腹血糖、PWT差异无统计学意义；注射STZ后，Dezocine组和Control组大鼠体重显著降低、空腹血糖明显升高、PWT显著降低（P0.05）。注射地佐辛后，与Control组相比，地佐辛组大鼠PWT在T2时间点显著升高（P0.05），在T3、T4时间点无明显差异（P0.05）；Control组和Dezocine组两组大鼠的体重、空腹血糖在各时间点均无明显差异（P0.05）。 2三组大鼠脊髓背角NMDA-2B受体的表达变化比较：与Normal组相比，Dezocine组和Control组两组大鼠NMDA-2B受体免疫阳性神经元细胞数量、mRNA含量在T2、T3、T4时间点显著升高（P0.05），与Control组大鼠相比，Dezocine组大鼠NMDA-2B受体免疫阳性神经元细胞数量、mRNA含量在T2时间点显著降低（P0.05），在T3、T4差异无统计学意义（P0.05）。在Dezocine组内NMDA-2B受体免疫阳性神经元细胞数量、mRNA含量T3时间点与T2比较显著升高（P0.05）。结论：地佐辛可以有效缓解糖尿病神经病理性疼痛大鼠的病理性痛；地佐辛可以抑制糖尿病神经病理性疼痛大鼠脊髓背角NMDA-2B受体表达上调。
[Abstract]:Objective: Diabetic peripheral neuropathy (DPN) pain is a type of neuropathic pain (NP), and DPN is one of the most common complications of diabetes. The morbidity of DPN is about 7.5% to 24%. The pathogenesis of DPN is very complicated. The central sensitization plays an important role in the production and maintenance of DPN, and the excitatory neurotransmitter glutamate and its receptor NMDA are involved in the occurrence of central sensitization. As a subunit of the NMDA receptor, NMDA-2B is especially important for the transmission of nociceptive signals. Therefore, the research on the NMDA receptor in recent years has been focused on the NMDA-2B subunit. As a novel and potent hybrid opioid receptor agonist-antagonist, Zorzin is characterized by strong analgesic effect and low adverse reaction, and has been widely used in clinic In this study, the effect of tramadol on DPN was found to be effective in the treatment of DPN, and the effect of DPN on DPN was not very clear. Type, and then give the ground Zusin to intervene. Changes in body weight, fasting blood glucose and 50% reduced foot response threshold (PWT) in three groups of rats were prepared by observing the normal group (Normal group), the diabetic neuropathic pain group (Control group), and the diabetic neuropathic pain model. Immunohistochemistry and RT-PCR were used to compare the expression of NMDA-2B receptor in the spinal dorsal horn of diabetic neuropathic pain rats. To study the effect of zocassin on the pain of diabetic patients with neuropathic pain and its potential role in the treatment of diabetic neuropathic pain Methods:80 healthy adult male SD rats, weighing 130 g ~ 150 g, were used in animal experiment of Hebei Medical University The model was prepared by randomly selecting 60 rats for intraperitoneal injection of STZ (50 mg/ kg). After a week of injection of STZ, the fasting blood glucose of 45 rats was 16.7 mol/ L (the success rate was 75%), and it was qualified. The PWT and PWT of the experimental rats were measured with von Frey Hairds two weeks after the injection of STZ for two weeks. Rat model. The remaining 20 rats were treated as normal control rats and the STZ abdomen was replaced with the same volume of saline. The rats were randomly divided into 2 groups,18 in each group: one group of Dzocine group and 2 weeks after the injection of STZ, and 2 weeks after the injection of STZ, the outer side of the thigh was injected intramuscularly by intramuscular injection of 2.52 mg/ kg, once a day, and the continuous muscle The meat was injected for 1 week; a group of diabetic neuropathic pain rats (Control group), with the same volume of physiological saline instead of Doxacin, the method was the same as above; in the normal group,18 groups were taken as the normal group (Normal group), and the same volume of physiological saline was used instead of the ground Zusin. The body weight, fasting blood glucose and PWT of each group were measured at 5 time points after injection of STZ (T0), the second week (T1), the third week (T2), the fifth week (T3) and the seventh week (T4), respectively. Three rats were used for immunohistochemical staining. After the anesthesia,4% paraformaldehyde solution was used for the fixation, and the spinal cord from the enlarged part of the thoracic waist of the rat was made into a wax block, and the immunohistochemical staining was performed to determine the immunopositive of the NMDA-2B receptor in the dorsal horn of the spinal cord of the rat. The number of nerve cells was measured; in the 3 rats, the spinal cord tissue was rapidly extracted at the enlargement of the thoracic and lumbar segment of the rat, and the m-2 B receptor was determined by means of the PT-PCR technique. RNA content. The analysis was performed using the SPSS17.0 statistical software, and the measurement data was level. The mean square standard deviation (x _% s) indicated that one-factor analysis of variance was used in the intra-group comparison, and t-test was used between the groups, and the difference between the groups was P0.05. difference Statistical significance. Results: The changes in body weight, fasting blood glucose and 50% contraction threshold of three groups of rats were compared with those of three groups: before the injection of STZ, the weight of three groups of rats, the fasting blood glucose and the PWT difference were not statistically significant; after the injection of STZ, the weight of the Dezocine group and the Control group in rats obviously, the fasting blood sugar is obviously increased, and P In the control group and Dezocine group, the weight of PWT in the control group and the Dezocine group was significantly higher than that of the control group (P0.05). There was no significant difference in the expression of NMDA-2B receptor in the spinal dorsal horn of the three groups (P0.05). Compared with the normal group, the number of NMDA-2B receptor-positive neurons in the two groups of the Dezocine group and the Control group was significantly higher than that of the normal group (P0.05). Results Compared with the control group, the number of NMDA-2B receptor-positive neurons in the Dezocine group was significantly lower at the time of T2 (P0.05), at the time of T3, T4. There was no significant difference in the expression of NMDA-2B receptor in Dezocine group (P0.05). Conclusion: Zusin can effectively relieve the pathological pain of diabetic neuropathic pain in rats.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R614;R-332

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