TIM-1、TIM-3在人慢性牙周炎牙龈组织中肥大细胞上表达的研究

发布时间：2018-01-05 11:17

本文关键词：TIM-1、TIM-3在人慢性牙周炎牙龈组织中肥大细胞上表达的研究　出处：《暨南大学》2015年硕士论文　论文类型：学位论文

【摘要】：背景:牙周炎(periodontitis)是指累及牙周支持组织的局部炎症性破坏性疾病。牙周炎已被医学界定论为继癌症、心脑血管疾病之后,威胁人类身体健康的第三大杀手,是导致牙齿脱落的主要原因。牙周炎以菌斑为始动因子,牙龈卟啉单胞菌(Porphyromonas gingivalis,Pg)、伴放线放线杆菌(Actinobacillus actinomycetemcomitans,Aa)以及福赛坦氏菌(Tannerella forsythensis,Tf)等革兰氏阴性厌氧菌大量堆积在牙龈边缘引起牙周组织的炎症反应。同时,牙周致病菌释放的脂多糖(lipopolysaccharide,LPS)、抗原和毒性因子等致病成分,激发宿主免疫反应引起机体免疫损伤,导致局部炎症细胞浸润并释放多种细胞因子,进一步造成宿主牙周组织的破坏及牙槽骨的吸收。多种免疫细胞及细胞因子参与牙周炎的免疫调节,宿主的易感性及牙周致病菌之间的相互作用决定了牙周炎的发生及进展。近年来认为,肥大细胞(mast cells,MCs)除了作为超敏反应的主要效应细胞外,MCs作为调节和效应性免疫细胞在适应性免疫反应的调节和固有免疫的建立中发挥重要作用,主要参与粘膜炎症、机体防御和组织修复等。MCs被激活后,可通过脱颗粒释放一系列介质,引起炎症反应或参与机体防御反应,发挥多种免疫功能。研究显示,在正常牙龈组织及牙周炎牙龈组织中均检测到MCs。研究表明,类胰蛋白酶(tryptase)阳性MCs在人慢性牙周炎牙龈组织中表达最为丰富,并随牙周炎的病变程度的增加而表达升高。T细胞免疫球蛋白粘蛋白(T cell immunoglobulin mucin domain,TIM)基因家族是T细胞表面的跨膜蛋白家族。TIM-1和TIM-3是TIM家族中的两个重要成员,分别在分化成熟的Th2细胞和Th1细胞上表达,参与Th细胞免疫反应的调控。目前tryptase-TIM-1、TIM-3双阳性MCs在人慢性牙周炎上的表达及功能尚不清楚,有关牙周炎牙龈组织tryptase-TIM-1、TIM-3双阳性MCs在牙周炎的发生和发展中的免疫调控作用尚未见报道。目的:本研究通过采集临床不同病变程度的牙周炎牙龈组织标本,经HE染色、甲苯胺蓝染色、免疫组织化学染色及免疫荧光双染色法,观察不同病变程度的牙周炎牙龈标本的炎症程度、MCs表达及脱颗粒情况,tryptase-TIM-1、TIM-3双阳性MCs的表达情况。通过对牙周炎牙龈组织的炎症程度等级评分、计算MCs平均阳性细胞率及脱颗粒率、tryptase-TIM-1、TIM-3双阳性MCs表达的密度,探讨tryptase-TIM-1、TIM-3双阳性MCs在牙周感染中的作用及其作用机制。研究结果将对后续以肥大细胞TIM-1、TIM-3为靶点的慢性牙周炎免疫防治提供理论基础和实验依据。方法:按纳入标准,将80例自愿参与本研究的受试者按慢性牙周炎分类标准分为4组:1)正常对照组20例;2)轻度慢性牙周炎组20例;3)中度慢性牙周炎组20例;4)重度慢性牙周炎组20例。牙龈标本置于10%中性福尔马林固定液中浸泡48 h以上,制作颊舌向5μm厚的牙龈组织连续切片。HE染色,于光学显微镜下观察各组牙龈组织的组织学变化;甲苯胺蓝染色及免疫组织化学染色,于光学显微镜下观察各组牙龈组织中MCs募集及脱颗粒情况,并计算MCs平均阳性细胞率及脱颗粒率;免疫荧光双染色,于荧光显微镜及共聚焦显微镜下观察tryptase-TIM-1、TIM-3双阳性MCs在牙龈组织中的表达,并计算tryptase-TIM-1、TIM-3双阳性MCs的密度(cells/mm2)。使用统计学软件SPSS 13.0对数据进行分析处理,采用Wilcoxon符号秩检验、Kruskal-Wallis H检验、Nemenyi法检验、单因素方差分析(one-way ANOVA)和Pearson相关分析,P0.05时认为差异有统计学意义。结果:1、组织学观察结果结果显示,4组牙龈组织切片炎症程度评分有差别,牙龈组织的炎症反应程度与慢性牙周炎的病变程度趋势一致:1)与正常对照组相比,各牙周炎组牙龈组织中的炎症细胞浸润明显增多,慢性牙周炎组炎症评分明显高于正常对照组(P0.01);2)中度慢性牙周炎组的炎症程度评分明显高于轻度慢性牙周炎组(P0.01);3)重度慢性牙周炎组的炎症程度评分明显高于轻度和中度牙周炎组(P0.01)。2、甲苯胺蓝染色及免疫组织化学染色结果结果显示,甲苯胺蓝染色与免疫组织化学染色的阳性细胞分布情况大致相同:1)与甲苯胺蓝染色相比,免疫组织化学染色中MCs平均阳性细胞率及脱颗粒率更高(P0.05),显色及脱颗粒现象更明显;2)慢性牙周炎组牙龈组织中MCs表达及脱颗粒现象比正常对照组明显增多,MCs平均阳性细胞率及脱颗粒率明显高于正常对照组(P0.01);3)中度慢性牙周炎组的MCs平均阳性细胞率及脱颗粒率明显高于轻度慢性牙周炎组(P0.01);4)重度慢性牙周炎组的MCs平均阳性细胞率及脱颗粒率明显高于轻、中度慢性牙周炎组(P0.01)。3、免疫荧光双染色结果1)慢性牙周炎组tryptase-TIM-1、TIM-3双阳性MCs的密度比正常对照组明显升高(P0.01);2)中度慢性牙周炎组的tryptase-TIM-1、TIM-3双阳性MCs的密度显著高于轻度慢性牙周炎组(P0.01);3)重度慢性牙周炎组的tryptase-TIM-1、TIM-3双阳性MCs的密度显著高于轻、中度慢性牙周炎组(P0.01)。4、MCs平均阳性细胞率和脱颗粒率、tryptase-TIM-1、TIM-3双阳性MCs与慢性牙周炎病变程度相关性分析结果经Pearson相关分析,各组MCs平均阳性细胞率和脱颗粒率、tryptase-TIM-1、TIM-3双阳性MCs密度均与慢性牙周炎的病变程度存在明显的正相关关系。结论:1、慢性牙周炎牙龈组织中MCs平均阳性细胞率和脱颗粒率随慢性牙周炎的病变程度加重而增加。2、慢性牙周炎牙龈组织中tryptase-TIM-1、TIM-3双阳性MCs的密度随慢性牙周炎的病变程度加重而增加。3、牙龈组织中MCs募集和脱颗粒可能参与牙周感染免疫反应的病理过程,在牙周疾病的发病和进程中发挥重要作用;肥大细胞TIM-1、TIM-3可能参与慢性牙周炎的炎症反应,在慢性牙周炎的发病及疾病进程中发挥关键作用。
[Abstract]:Background: periodontitis (periodontitis) is a devastating disease involving the periodontal support tissue local inflammation. Periodontitis has been inconclusive as medicine after cancer, cardiovascular diseases, threats to the third major killer of human health, is a major cause of tooth loss. The plaque is the initiating factor of periodontitis, Porphyromonas gingivalis (Porphyromonas gingivalis, Pg), Actinobacillus actinomycetemcomitans (Actinobacillus actinomycetemcomitans Aa) and Forseth Staw S (Tannerella forsythensis, Tf) bacteria such as gram negative anaerobic bacteria mass accumulation caused by periodontal tissue inflammation in the gingival margin. At the same time, periodontal bacteria release induced by lipopolysaccharide (lipopolysaccharide, LPS). Antigen and virulence factors of pathogenic components, stimulate the host immune response caused by immune injury, causing local infiltration of inflammatory cells and release cytokines, further made To destroy the host periodontal tissue and alveolar bone absorption. A variety of immune cells and immune cytokines involved in the regulation of periodontitis, host susceptibility and interaction between periodontal pathogens determines the incidence and progression of periodontitis. In recent years, mast cells (mast cells, MCs) in addition to the main effect as hypersensitivity the outside of the cell, MCs as regulatory and effector immune cells play an important role in the establishment and regulation of innate immunity in the adaptive immune response, mainly involved in mucosal inflammation, immune defense and tissue repair after.MCs is activated by degranulation to release a series of medium, cause inflammation or immune reaction in host defense. A variety of functions. The results show that in normal gingival tissue and periodontal tissues were detected in MCs. studies show that tryptase (tryptase) positive MCs in patients with chronic periodontitis The most abundant expression in gingival tissue, and increased with the severity of periodontitis and increased expression of.T cell immunoglobulin mucin (T cell immunoglobulin mucin domain, TIM T) gene family is a cell surface transmembrane protein family.TIM-1 and TIM-3 are two important members in the TIM family, were expressed in Th2 cells and the differentiation of Th1 cells, is involved in regulation of Th cell immune response. At tryptase-TIM-1, the expression and function of TIM-3 double positive MCs in human chronic periodontitis on is not clear, the periodontal tissues tryptase-TIM-1, immune regulation of TIM-3 double positive MCs in the occurrence and development of periodontitis has not been reported. Objective: this through the study of the collection of the clinical severity of periodontitis gingival tissue specimens by HE staining, toluidine blue staining, immunohistochemical staining and double immunofluorescence staining, observe The degree of inflammation with the severity of periodontitis gingival tissues, the expression of MCs and degranulation, tryptase-TIM-1, expression of TIM-3 double positive MCs. The periodontal tissue inflammation score, MCs calculated the average rate of positive cells and degranulation rate, tryptase-TIM-1 expression, TIM-3 double positive MCs density of tryptase-TIM-1, TIM-3 double positive MCs in periodontal infection of the role and its mechanism. The results of the follow-up to the mast cell TIM-1, TIM-3 provide a theoretical basis and experimental basis for targeted immune prevention of chronic periodontitis. Methods: according to the inclusion criteria, subjects 80 volunteers participated in this study according to the classification standard of chronic periodontitis 4 group: 1) and 20 cases of normal control group; 2) mild chronic periodontitis group 20 cases; 3) moderate chronic periodontitis group 20 cases); 4 patients with severe chronic periodontitis and 20 cases of gingival specimens were placed in 10% neutral Holmes. Ma Lin fixed buffer for 48 h or more, making the buccolingual 5 m thick continuous gingival tissue slices were stained with.HE, were observed by microscope and the histological changes of gingival tissue in each group; toluidine blue staining and immunohistochemical staining were observed under optical microscope in gingival tissue of MCs recruitment and degranulation, and calculate the average MCs positive cell rate and degranulation rate; double immunofluorescent staining, fluorescent microscopy and confocal microscope tryptase-TIM-1, TIM-3 double positive expression of MCs in gingival tissue, and calculate the tryptase-TIM-1, TIM-3 double positive MCs density (cells/mm2). 13 on the use of statistical software SPSS data analysis and processing, using Wilcoxon signed rank test, Kruskal-Wallis H test, Nemenyi test, single factor analysis of variance (one-way ANOVA) and Pearson correlation analysis, P0.05 believes that there are significant differences. Results: 1. Histological observation results showed that 4 groups of gingival tissue slices inflammation score difference, consistent lesions trend degree of inflammatory reaction of gingival tissue with chronic periodontitis: 1) compared with the normal control group, the inflammatory cell infiltration in periodontitis gingival tissue increased significantly, chronic periodontitis group inflammation score significantly higher than the normal control group (P0.01); 2) moderate chronic periodontitis group the inflammation score was significantly higher than that of mild chronic periodontitis group (P0.01); 3) severe chronic periodontitis group inflammation score was significantly higher than that of mild and moderate periodontitis group (P0.01).2, toluidine blue staining and immunohistochemical staining showed that toluidine blue staining immunohistochemical staining positive cells and the distribution of roughly the same: 1) compared with toluidine blue staining, immunohistochemical staining of MCs positive cells and the average rate of The degranulation rate is higher (P0.05), color and degranulation phenomenon is more obvious; 2) and degranulation of the expression of MCs in gingival tissue of chronic periodontitis group was significantly increased than the normal control group and normal control group, the rate of degranulation was significantly higher than the average MCs positive cells (P0.01); 3) the average rate of MCs positive cells moderate chronic periodontitis group and degranulation rate was significantly higher than that of mild chronic periodontitis group (P0.01); 4) severe chronic periodontitis group MCs average rate of positive cells and degranulation rate was significantly higher than that of mild, moderate chronic periodontitis group (P0.01.3), double immunofluorescence staining results in 1 chronic periodontitis group) tryptase-TIM-1, TIM-3 double positive MCs the density was significantly higher than the normal control group (P0.01); 2) moderate chronic periodontitis group tryptase-TIM-1, TIM-3 double positive MCs density was significantly higher than that of mild chronic periodontitis group (P0.01); 3) severe chronic periodontitis group tryptase-TIM-1, TIM-3 The positive MCs density was significantly higher than that of light, moderate chronic periodontitis group (P0.01.4 MCs), the average rate of positive cells and degranulation rate, tryptase-TIM-1, MCs and TIM-3 double positive chronic periodontitis lesions correlation analysis results by Pearson correlation analysis, MCs was the average rate of positive cells and degranulation rate, tryptase-TIM-1, there is a significant positive correlation the lesions were chronic periodontitis and TIM-3 double positive MCs density. Conclusion: 1. The gingival tissues of patients with chronic periodontitis MCs average positive cell rate and degranulation rate with the severity of chronic periodontitis aggravates the increase of.2, tryptase-TIM-1 in gingival tissues of patients with chronic periodontitis and chronic periodontitis lesions with TIM-3 double positive MCs density increased the increase of.3, MCs in gingival tissue recruitment and degranulation of periodontal infection may be involved in the immune response of the pathological process in the pathogenesis of periodontal disease and in the course of play The mast cell TIM-1, TIM-3 may participate in the inflammatory response of chronic periodontitis, and play a key role in the pathogenesis of chronic periodontitis and the process of disease.

【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R781.4

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