钠通道亚型Nav1.3在大鼠实验性牙髓炎组织中的表达

发布时间：2018-04-28 10:40

本文选题：电压门控钠离子通道 + Nav1.3　；参考：《安徽医科大学》2014年硕士论文

【摘要】：目的电压门控钠通道亚型Nav1.3属于河豚毒素敏感型（TTX-S）钠通道，Nav1.3主要表达于啮齿动物胚胎脑组织，可快速激活和快速失活Na+电流，，对动作电位有显著调节作用，在炎症刺激和神经元损伤后重新表达并出现异位。牙髓组织的感觉功能由疼痛感受器来支配，特定的神经纤维构成疼痛感受器。正常情况下，适宜外界刺激不会引起牙髓组织的不适反应，但如继发牙髓炎等牙髓炎性病变时，牙髓组织中的疼痛感受器会产生痛觉反应。本实验通过构建实验性大鼠牙髓炎模型，对比分析钠通道Nav1.3在正常大鼠牙髓组织及轻度、中度、重度牙髓炎大鼠牙髓组织中表达的不同，探讨钠通道Nav1.3表达量与牙髓炎所致炎性疼痛之间的关系，有助于临床上调整牙痛的治疗靶点，有助于寻找优于传统局部麻醉剂的有效缓解牙痛的新方法。方法将16只大鼠随机分为对照组、实验1d组、3d组、5d组，每组4只。大肠杆菌内毒素(LPS)诱导大鼠牙髓炎后，在伤后1、3、5d处死，通过对构造模型后对大鼠的动物行为学观察，通过HE染色检测对照组及实验1d、3d、5d组牙髓组织的病理状况，利用反转录聚合酶链（RT-PCR）法检测对照组及实验1d、3d、5d组牙髓组织中TNF-α mRNA水平的表达量，判断牙髓炎严重程度与炎性疼痛之间的关系；利用免疫组织化学技术检测Nav1.3在正常大鼠及牙髓炎大鼠牙髓组织中的定性表达；Western blot方法检测并比较Nav1.3在正常大鼠及牙髓炎大鼠牙髓组织中的蛋白相对表达量。结果1.动物行为学观察结果表明，正常组大鼠饮食二便正常，精神状态较佳；实验组大鼠在造模后出现反复舔伤口，不敢咬食物，烦躁不安，尖叫不止，体重降低等表现；2.HE染色结果表明：正常组的组织结构正常，无病理性改变；随着牙髓炎严重程度的增加，实验组的组织中逐渐出现中性粒细胞浸润，成牙本质细胞排列紊乱，血管扩张充血等病理性改变；3. RT-PCR法检测TNF-α结果表明，与对照组相比，实验1d组、3d组、5d组的TNF-α mRNA水平的表达量随着牙髓炎严重程度的增加而升高（P0.05）；4.免疫组织化学染色结果表明：正常组、实验1d组、3d组、5d组的平均光密度分别为：0.2631±0.0609、0.3628±0.0610、0.5249±0.1815、0.6354±0.2160，实验组与对照组相比，差异有统计学意义(P＜0.05)；5. Western blot分析结果表明：正常组、实验1d组、3d组、5d组的Nav1.3的相对表达强度分别为：0.320±0.041、0.551±0.038、0.983±0.027、1.142±0.019，实验组与对照组相比，差异有统计学意义(P＜0.05)。结论本研究证实大鼠牙髓组织中存在Nav1.3的表达，牙髓炎所致的炎性疼痛可导致Nav1.3表达显著上调，这可能是炎性疼痛发生时神经元细胞膜上钠通道功能异常的分子学基础之一。钠通道Nav1.3与牙髓炎痛觉致敏之间可能存在潜在关联，牙髓炎导致炎性疼痛时，Nav1.3表达上调很可能参与了成牙本质细胞的兴奋性反应，可进一步推测临床上研制Nav1.3抑制剂，可能有助于临床上调整牙痛的治疗靶点。
[Abstract]:Objective voltage gated sodium channel subtype Nav1.3 belongs to tetrodotoxin sensitive (TTX-S) sodium channel. Nav1.3 is mainly expressed in rodent embryonic brain tissue. It can quickly activate and quickly deactivate Na+ current. It has a significant regulating action on action potential. It can be re expressed and ectopic after inflammatory stimulation and neuron injury. The sensory work of dental pulp tissue It can be controlled by pain receptors, specific nerve fibers form a pain receptor. Under normal circumstances, appropriate external stimuli do not cause discomfort in dental pulp tissue, but when pulpitis secondary to pulpitis, such as secondary pulpitis, the pain receptor in the pulp tissue will produce pain response. This experiment was made by constructing an experimental rat model of pulpitis. The expression of sodium channel Nav1.3 in pulp tissue of normal rats and mild, moderate, and severe pulpitis rats was compared and analyzed. The relationship between the expression of sodium channel Nav1.3 and inflammatory pain caused by pulpitis was discussed, which could help to adjust the therapeutic target of toothache, and help to find better than the traditional local anesthetic. A new way to relieve toothache.
Methods 16 rats were randomly divided into control group, experimental group 1D, group 3D and group 5D, 4 rats in each group. After Escherichia Coli Endotoxin (LPS) induced rat pulpitis, 1,3,5d was executed after the injury. After the structural model, the animal behavior of rats was observed. HE staining was used to detect the pathological condition of the control group and the experimental 1D, 3D, 5D group of the pulp tissue, and the reversal of the pathological condition of the pulp tissue in the group of 1D, 3D, and 5D. RT-PCR method was used to detect the expression of TNF- alpha mRNA in the pulp tissues of the control group and the experimental group of 1D, 3D and 5D, to determine the relationship between the severity of pulpitis and the inflammatory pain, and to detect the qualitative expression of Nav1.3 in the dental pulp tissues of normal rats and pulpitis rats by immunohistochemical technique and the detection of Western blot method. The relative expression of Nav1.3 protein in dental pulp tissue of normal rats and pulpitis rats was compared.
Results the results of 1. animal behavior observation showed that the diet of normal rats was normal and the mental state was better. The rats in the experimental group appeared repeatedly licking the wound after making the model, did not dare to bite the food, was restless, screamed, and lost weight. The results of 2.HE staining showed that the structure of the normal group was normal, without pathological changes; with the teeth. The severity of myeloid inflammation increased, the tissue of the experimental group gradually appeared neutrophils infiltration, odontoblasts were arranged disorder, vascular dilatation and congestion and other pathological changes, and the results of TNF- alpha detected by 3. RT-PCR method showed that the expression of TNF- alpha mRNA in experimental group 1D, 3D group and 5D group increased with the increase of the severity of pulpitis. The results of 4. immunohistochemical staining showed that the average light density of the normal group, the experimental group 1D, the 3D group and the 5D group were respectively: 0.2631 + 0.0609,0.3628 + 0.0610,0.5249 + 0.2160, and the experimental group was statistically significant (P < 0.05) compared with the control group (P < 0.05), and 5. Western blot analysis showed that the normal group was normal, The relative expression intensity of Nav1.3 in group 1D, group 3D and 5D group was 0.320 + 0.041,0.551 + 0.038,0.983 + 0.019 respectively. The difference was statistically significant (P < 0.05) compared with the control group (P < 0.05).
Conclusion the present study confirmed the expression of Nav1.3 in the pulp tissue of rats, and the inflammatory pain caused by pulpitis can lead to a significant up-regulated expression of Nav1.3. This may be one of the sub molecular basis of the abnormal sodium channel function on the membrane of neuronal cells during the occurrence of inflammatory pain. There may be a potential association between sodium channel Nav1.3 and the sensitization of dental myelomyctis. When pulpitis causes inflammatory pain, the up-regulated expression of Nav1.3 is likely to be involved in the excitatory response of odontoblasts. It is possible to further speculate that the clinical development of Nav1.3 inhibitors may contribute to the therapeutic targets for the clinical adjustment of toothache.

【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R781.31

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