【作者】 刘鑫；

【导师】 向学熔；

【作者基本信息】 重庆医科大学 ， 口腔临床医学， 2008， 硕士

【摘要】 背景:牙髓中细胞外基质(extracellular matrix, ECM)是由牙髓成纤维细胞和成牙本质细胞等合成并分泌到细胞外的生物大分子所构成的网络状物质,分布于细胞之间或细胞表面,其化学成分的动态变化是一个窗口,通过它可以窥测牙髓细胞的代谢活性和功能。基质金属蛋白酶(matrix metalloproteinases, MMPs)在牙髓组织细胞外基质代谢过程中发挥着关键性作用,是牙髓病过程中组织损伤机制中的重要因子,其基因表达、蛋白质合成以及活性的表达受到多种因素的调节,对MMPs的研究可以为牙髓病的治疗提供新思路。正畸力适宜时牙根尖部血管受轻压,牙髓组织可发生轻度充血、对温度的变化敏感,有时可出现牙髓活力下降,但并不出现显著的牙髓炎症改变,对正畸力作用下牙髓中MMPs的活性及表达的研究目前依然比较少。因此,进一步研究MMPs在不同情况下的牙髓中的表达,受哪些因素影响,如何影响就显得很有必要。目的:采用免疫组织化学方法,研究人正常牙髓、炎症牙髓和接受正畸力作用影响的牙髓中MMP-2和MMP-9的表达,探讨MMPs激活在牙髓炎性反应的发病机制中发挥的作用,为探索有效的MMPs对抗机制,阻止蛋白酶对ECM的有害作用,以及为从阻止基质的降解和缓解炎性疼痛方面减轻牙髓炎性疼痛提供一定的实验依据。材料与方法:1、选取包括因正畸治疗需要而拔除的健康前磨牙和因需要而拔除的健康第三磨牙共20颗。对标本进行处理后制成切片,采用链霉素抗生素蛋白—过氧化酶免疫组化法( SP法),对其中MMP-2和MMP-9的表达进行图像分析和半定量分析。2、选取包括因正畸治疗需要而拔除的健康前磨牙和因需要而拔除的健康第三磨牙共20颗,分为正常组;选取临床诊断有牙髓炎症且无保留价值的第3磨牙20例,分为炎症组。对标本进行处理后制成切片,采用SP法,对其中MMP-2和MMP-9的表达进行图像分析和半定量分析。3、选取接受正畸治疗的患者10名(男女各5名),于正畸治疗前确定应拔除的正畸牙,对其一侧选用常规正畸加力,力值约每牙20-25克,另一侧健康同名牙不施加任何正畸力,作为同源对照,加力一天后至七天内拔除该正畸牙,各20颗,共40颗,分为加力组和对照组。选取临床诊断有牙髓炎症且无保留价值的第3磨牙20例,分为炎症组。对标本进行处理后制成切片,采用SP法,对其中MMP-2和MMP-9的表达进行图像分析和半定量分析。结果:1、正常牙髓中,主要在成牙本质细胞和成纤维细胞的胞质可见MMP-2和MMP-9的表达,其表达呈弱阳性,牙髓中央区域表达弱于外周,而MMP-9表达水平较MMP-2高。半定量积分分析:MMP-2有18例弱阳性表达,2例阴性表达,阳性构成比为90.00%;MMP-9有19例阳性表达,1例中度阳性表达,阳性构成比为100.00%。2、正常和炎症牙髓均可见MMP-2、MMP-9的表达。正常牙髓组织内MMP-2、MMP-9阳性细胞数较炎症组织少。在炎症牙髓组织,成牙本质细胞、牙髓成纤维细胞和血管内皮细胞可见MMP-2、MMP-9表达,炎细胞浸润区表达呈阳性,MMP-9表达仍强于MMP-2。半定量积分分析:炎症组牙髓中MMP-2和MMP-9表达增强例数分别为18例和20例,与正常组差异具有统计学意义(P<0.05)。3、对照组、加力组和炎症组牙髓均可见MMP-2、MMP-9的表达。正常牙髓组织内MMP-2、MMP-9阳性细胞数较加力组和炎症组的牙髓组织少。在接受正畸力的牙髓组织,成牙本质细胞和牙髓成纤维细胞可见MMP-2、MMP-9表达,从阳性染色强度来看,在牙髓中央区域表达弱于外周。MMP-9表达强于MMP-2 ,并且两者表达均明显强于正常牙髓组织中的表达,且弱于炎症牙髓组织。半定量积分分析:加力组牙髓中MMP-2和MMP-9为表达增强,并且两者表达强于同源对照正常的牙髓,但弱于炎症牙髓,三组差异具有统计学意义(P<0.05)。结论:MMP-2和MMP-9在人牙髓中主要表达于成牙本质细胞和牙髓成纤维细胞的胞质中。生理条件下,MMP-2和MMP-9的水平较低,其与TIMPs的平衡维持着牙髓组织的健康。当牙髓受到炎性刺激后,MMP-2和MMP-9的表达水平增强,两者的表达水平可以反映出炎症的水平和进程。由于MMPs是牙髓病理过程中组织损伤机制中的重要因子,对MMPs的研究可以为牙髓病的治疗提供新思路。在接受正畸治疗时,由于受正畸力影响,牙髓处于应激状态,MMP-2和MMP-9表达增强,虽未引起明显牙髓炎症情况,但也应引起口腔医师的高度重视,以期获得最佳治疗方案,提高治疗效率,并为患者提供更好的医疗服务。更多还原

【Abstract】 Background:Extracellular matrix(ECM) is a substance made of bio-macromolecules which is synthetized and excreted to extracellular by odontoblasts and fibroblasts of dental pulp.It spreads among the cells and the surface of cells.Its chemical composition is a port to explore the metabolic activities and functions of pulp cells.Matirx metalloproteinases(MMPs) produces key marked effects during metabolic processes of pulp tissues ECM.It’s the important factor of tissue damage mechanism in pulposis process.Its genetic expression, protein synthesis and activity expression are subjected to accommodations of many factors.It can provide new train of thoughts for healing pulposis by investigating the MMPs.While the orthodontic forces are convenience,the blood vessels of root tip are pressed slightly,the pulp tissues come about to hyperemia in slight degree and sensitiveness to the variation of temperature.Sometimes,the pulp vitality decreases.Nevertheless,there is no remarkable change of endodontitis. For the time being,the research for the expressions of MMPs in dental pulp effected by orthodontic forces is still a gap.It is necessary to explore the MMPs’expressions of dental pulps in different conditions further,what factors influence them,and how the influences go.Objective:The method of immunohistochemistry was adopted.Studied the expressions of MMP-2 and MMP-9 in human dental pulps while they are normal, inflamed and under the effections of orthodontic forces.Probed the roles of MMPs activated in pathogenesis for inflammatory reaction of dental pulp.This study can provide important experimental evidence for effective antagonized mechanisms to MMPs,for preventing the deleterious effects of MMPs done to ECM,and for mitigating inflamed pains of dental pulps by preventing ECM degradation and reliefing inflamed pains.Materials and Methods:1.Selected 20 teeth which were healthy premolares extracted because of orthodontic treatments and healthy third molares extracted for needs. Made microtome sections after essays dealing.Used immunohistochemical streptavidin peroxidase method(SP method), analysed the expressions of MMP-2 and MMP-9 by analysing images and half-quantitative analysis.2. Selected 20 teeth which were healthy premolares extracted because of orthodontic treatments and healthy third molares extracted for needs as nomal group.Selected 20 third molares that clinical diagnoses were endodontitis and had no worth remaining as inflamed group. Used immunohistochemical streptavidin peroxidase method(SP method), analysed the expressions of MMP-2 and MMP-9 by analysing images and half-quantitative analysis.3. Selected 10 orthodontic patients(5 male and 5 female) ,decided which orthodontic teeth should be extracted before orthodontic treatments had started.Chose one side of them,and adopted routine orthodontic forces about 20-25g.Extracted the teeth within 1 to 7 days after orthodontic forces had been used.There were 20 teeth as under-forces group. The other side of the pre-extracted teeth did not use any orthodontic forces.There are also 20 teeth as homologizd control group. Selected 20 third molares that clinical diagnoses were endodontitis and had no worth remaining as inflamed group.Used immunohistochemical streptavidin peroxidase method(SP method), analysed the expressions of MMP-2 and MMP-9 by analysing images and half-quantitative analysis.Results:1.In healthy pulps,MMP-2 and MMP-9 mainly expressed in cytoplasts of odontoblasts and fibroblasts.The expressions were weakly positive.The central areas expressed more manifest than the peripheral one.The expression level of MMP-9 was higher than the MMP-2. Half-quantitative analysis:the positive constituent ratio of MMP-2 was 90.00%,and of MMP-9 was 100.00%. 2.There were MMP-2 and MMP-9 expressions in healthy and inflamed pulps.In healthy pulpal tissues,there were less positive cells with MMP-2 and MMP-9 than in inflamed pulps.In inflamed pulps, there were expressions of MMP-2 and MMP-9 in odontoblasts,fibroblasts and vascular endothelial cells.The areas of inflamed cells were positively expressed. The expression level of MMP-9 was also higher than the MMP-2. Half- quantitative analysis:in inflamed pulps,the expressions of MMP-2 and MMP-9 were heightened.Compared with normal group,the differences had statistical significance(P<0.05).3.There were expressions of MMP-2 and MMP-9 in control,under- forces and inflamed groups. In control pulpal tissues,there were less positive cells with MMP-2 and MMP-9 than in under-forces pulps and inflamed pulps.In under-forces pulpal tissues, MMP-2 and MMP-9 mainly expressed in odontoblasts and fibroblasts.Analyzed with the magnitude of staining, the central areas expressed more manifest than the peripheral one. The expression level of MMP-9 was higher than the MMP-2. In under- forces pulps,the expressions of MMP-2 and MMP-9 were manifestly stronger than control one,but weaker than the inflamed one.Half- quantitative analysis: in under-forces pulps,the expressions of MMP-2 and MMP-9 were heighten.The expressions:control<under-forces<inflamed pulps.The differences among the three groups had statistical significance(P<0.05). Conclusions:MMP-2 and MMP-9 mainly expressed in cytoplasts of odontoblasts and fibroblasts in human dental pulps.In physiological conditions,the expression levels of MMP-2 and MMP-9 are low,and sustain the health of pulpal tissues by the balance between MMPs and TIMPs.After stimulated by inflammations, the expressions of MMP-2 and MMP-9 are heightened. It can show the inflamed level and processes.Because of high-expressions of MMPs in inflamed pulps,they are important factors of tissue damage mechanisms during the pathological processes of dental pulps. It can provide new train of thoughts for healing pulposis by investigating the MMPs.During orthodontic treatments, effected by the orthodontic forces, the dental pulps are in stringent state. The expressions of MMP-2 and MMP-9 are heightened.Although it does not cause the pulpitis,the dentists should pay more attention to this fact. The orthodontic treatments are based on intimately knowing the influence of orthodontic forces on patients’organisms and using biomechanic concepts correctly.Only by this means can we increase the efficiency and design the best proposal of therapy.更多还原