【作者】 林彦菊；

【导师】 张晓芳；

【作者基本信息】 中国医科大学 ， 口腔临床医学， 2010， 硕士

【摘要】 目的诱导型一氧化氮合酶(inducible nitric oxide synthase, iNOS)是机体合成NO的最主要限速因子之一,可以使体内产生高出生理浓度的一氧化氮(nitric oxide,NO)。而NO是细胞信息传递的重要调节因子,犹如一把双刃剑广泛参与了机体的生理及病理过程。牙齿在受各种刺激如损伤、根折、正畸力作用及可逆性牙髓炎时可引起牙髓内一些细胞分泌各种多肽类生长因子,从而增加血管通透性,促进血管生成。血管生成是牙本质成功修复的前提,血管内皮生长因子(vascular endothelial growth factor, VEGF)就是其中一种重要的血管生成因子。牙髓受到各种刺激如创伤等可以使牙髓组织产生一系列的反应,具有一定的抵御刺激、进行自身修复反应的能力,但是牙髓修复的机制尚不明确。本研究旨在探讨iNOS和VEGF在大鼠牙髓机械损伤后修复过程中的表达和二者之间的关系,及不同盖髓剂直接盖髓后iNOS表达变化和分布规律,为iNOS在牙髓损伤修复中的作用机制提供实验依据。材料和方法制备大鼠牙髓机械损伤模型及氢氧化钙(calcium hydroxide, CH)和矿物三氧化物凝聚体(mineral trioxide aggregate, MTA)直接盖髓模型,分别于处理后即刻、1d、3d、7d、14d、21d、28d用4%多聚甲醛心脏灌流固定、取材、脱钙后常规石蜡包埋,制备切片,采用免疫组织化学染色(SABC法)观察牙髓机械损伤组牙髓的病理变化及iNOS和VEGF表达情况,和盖髓组iNOS的表达变化,用显微照相系统及图像分析软件测定各组标本阳性染色的平均光密度值(optical density,OD)。利用SPSS13.0统计软件对所得数据进行单因素方差分析(ANOVA)、Pearson相关分析及配对t-检验,P<0.05时有显著性差异。结果1、在正常牙髓组织中iNOS染色呈阴性,开髓即刻牙髓中iNOS出现低水平表达,3d组牙髓iNOS出现强阳性表达,损伤7d后iNOS表达开始减弱,损伤21d后接近正常水平。表达主要位于中性粒细胞,成纤维细胞,成牙本质细胞和少数血管内皮细胞。损伤后1d、3d、7d、14d iNOS表达强度与正常对照有差异。2、正常牙髓VEGF在血管内皮细胞染色呈强阳性,成牙本质细胞染色弱阳性或无着色。牙髓损伤后,VEGF表达强度呈现先增加后降低的趋势,3d时表达最强,7d略有降低,后逐渐降低至28d时接近正常水平。表达主要位于血管内皮细胞,成纤维细胞,中性粒细胞,部分成牙本质细胞。损伤后Id、3d、7d、14d VEGF表达强度与正常对照有差异。3、Pearson相关分析显示,iNOS与VEGF在牙髓损伤修复中的表达呈正相关关系(r=0.817,P<0.05)。4、两盖髓组即刻牙髓中iNOS出现低水平表达并逐渐增强,3d时达到高峰,7d后表达开始减弱,21-28d接近正常水平。MTA直接盖髓同一时点牙髓炎症程度较CH组低,修复性牙本质形成较CH组多,CH盖髓后3diNOS表达显著强于MTA组。结论1、大鼠牙髓损伤后,iNOS和VEGF的表达均呈先增高后降低的趋势,二者的表达变化与牙髓早期炎症状态和后期修复程度有关,iNOS和VEGF的表达强度具有正相关性。2、CH直接盖髓炎症及坏死程度较MTA组重,3d时iNOS显著高于MTA组,MTA可能通过某种途径降低牙髓iNOS活性,使牙髓炎症程度低于CH组,盖髓剂不同对牙髓修复程度和iNOS的表达强度有影响。更多还原

【Abstract】 ObjectiveInducible nitric oxide synthase (iNOS) is one of the most important limiting factor about the synthesis of nitric oxide (NO) in body, which can make the body to produce higher concentration of NO than physiological[1]. And NO is an important regulator in the process of cell information transmission, which extensively involved in the process of physiological and pathological like a double-edged sword[2].After subject to various incentives, such as injury, root fracture, orthodontic force and reversible pulpitis, some of the pulp cells may secrete a variety of peptide growth factors, which can increase vascular permeability and promote angiogenesis. Angiogenesis is a prerequisite for successfully repair of dentin, and vascular endothelial growth factor (VEGF) is one of an important vascular endothelial growth factors.When subject to various incentives, such as trauma, pulp can produce a series of reaction, including againsting stimulus and carrying out self-repair capacity. But the mechanism of pulp repair is not clear. This study aimed to explore the expression of iNOS and VEGF in rat dental pulp after injury, reveal the relationship between the two, and show the different distribution of iNOS expression after direct pulp capping with different pulp capping agent, in order to understand the role of iNOS in the process of dental pulp injury and repair, and find out the relationship between iNOS and VEGF.Materials and MethdodsPreparation of rat dental pulp mechanical injury model and direct pulp capping model with CH (calcium hydroxide) and MTA (mineral trioxide aggregate). The rats will be executed at immediately, 1d,3d,7d,14d,21d and 28d, heart perfusion fixed with 4% paraformaldehyde, drawn, decalcification, embedded in paraffin, prepare sections, and to observe the pathological changes and the expression of iNOS and VEGF in the group of mechanical injure, and the expression of iNOS in capping groups after immunohistochemistry (SABC). Software for image analysis was used to test the optical density (OD) of each Specimen. The data were analyzed by one-way ANOVA, pearson correlation analysis and paired t-test with SPSS 13.0, P<0.05 was considered statistically significant.Results1、In normal pulp, the stain of iNOS was negative; immediately after pulp injury was low-level expression while 3days was strong positive; after 7 days the stain became less positive gradually till 21 days approach to normal. The expression mainly located in neutrophils, fibroblasts, odontoblasts, and a few endothelial cells. The expression intensity of iNOS was different from normal controls after injury 1d,3d,7d,14d.2、The VEGF stained strongly positive, in vascular endothelial cells, weakly positive or no color in odontoblasts in normal pulp. After pulp injury, the staining of VEGF was markedly increased and then decreased, the strongest expression was at3 days,7days slightly decreased, and reduced to near normal levels at 28day. The expression mainly located in vascular endothelial cells, fibroblasts, neutrophils and some of the odontoblast. The expression intensity of VEGF was different from normal controls after injury 1d,3d,7d,14d.3、Pearson correlation analysis showed that iNOS and VEGF were positive correlation in the dental pulp injury and repair (r=0.817, P<0.05).4、In the two pulp capping groups, iNOS expression was weakly positive at immediately, while 3days was the most strongest positive; after 7 days the stain became less positive gradually till 21-28 days approach to normal. The degree of pulpitis in the group of MTA was lower than the CH, and reparative dentin was more than the CH group. The expression of iNOS in the group of CH was significantly higher than MTA, 3days after direct pulp capping.Conclusions1、In the process of rat dental pulp injury and repair, the expression of iNOS and VEGF expression was increased at first and then decreased, which has some relationship with the early inflammation and late repair degree and the expression of iNOS was correlated with VEGF.2、After direct pulp capping with CH, the degree of pulp inflammation and necrosis was worse than MTA, the expression of iNOS was significantly higher than MTA at 3 days.Maybe MTA can reduce the activity of iNOS in pulp, so that the degree of inflammation lower than the group of CH. The kind of pulp capping agents was impact on the pulp repairation and the expression of iNOS.更多还原