【作者】 沈静燕；

【导师】 刘士有； 邹朝晖；

【作者基本信息】 天津医科大学 ， 口腔临床医学， 2007， 硕士

【摘要】 目的：通过建立LPS诱导性大鼠牙髓炎模型，应用免疫组织化学的方法，观察LPS受体CD14、TLR4在LPS诱导的大鼠实验性牙髓炎模型中的定位情况和不同阶段的表达，并对其进行半定量分析，探讨CD14、TLR4在牙髓炎发生、发展中的作用，为阐明LPS在牙髓炎中的致病机理提供动物实验依据。方法：选用56只24周龄的雄性SD大鼠，随机分成7组，即1组正常组和6组牙髓炎组，每组8只。牙髓炎组的每只大鼠两侧的上颌第一磨牙分别设为实验组及对照组。实验组牙髓暴露后用LPS处理，对照组用生理盐水处理。分别于术后1天、3天、7天、2周、3周、4周行4％多聚甲醛心内灌注处死。处死后立即取下两侧上颌第一磨牙连同部分颌骨，经固定、脱钙、包埋后，作5μm厚的连续切片。HE染色观察牙髓组织经LPS、生理盐水处理后的组织病理学反应。免疫组织化学方法观察CD14、TLR4的定位及表达的动态变化。使用显微照像系统及Image pro plus5图像分析软件分别对牙髓组织中CD14和TLR4染色阳性部位进行灰度值测定，测得结果的平均值作为CD14和TLR4含量的最终结果。采用单因素方差分析(ONE-way ANOVA)，SNK-q检验及t检验进行统计学处理，P＜0.05时差异具有统计学意义。结果：LPS实验组牙髓存活长达4周以上，HE染色观察到了LPS对牙髓组织的刺激损伤和牙髓组织对LPS入侵的免疫防御以及自身修复的全过程。CD14在正常牙髓组织中，除了成牙本质细胞层的部分细胞呈阳性表达外，未见其他细胞呈CD14阳性表达；炎症牙髓组织中，炎性细胞(主要是单核／巨噬细胞、中性粒细胞)呈CD14强阳性着色。图像结果分析显示：正常组与LPS各炎症组间差异均有统计学意义(P＜0.05)；LPS各炎症组组间除了3周组与4周组两者间差异无统计学意义外(P＞0.05)，其余各组间差异均有统计学意义(P＜0.05)；1、3、7天时LPS组与NS组相比较，1天组两者之间差异无统计学意义(P＞0.05)，3、7天组两者差异均具有统计学意义(P＜0.05)。TLR4主要在血管内皮细胞、成牙本质细胞及炎细胞中表达。图像分析结果显示：正常组与LPS各炎症组相比，除了与2周组之间平均灰度值差异无统计学意义外(P＞0.05)，与其余各组均有统计学差异(P＜0.05)；LPS炎症各组之间比较，除了1天组与3天组之间差异无统计学意义外(P＞0.05)，其余各组之间差异均有统计学意义(P＜0.05)；1、3、7天时LPS组与NS组相比较，各时间段均显示两者之间差异有统计学意义(P＜0.05)，同一时间段LPS组的平均灰度值低于NS组。结论：①LPS诱导的大鼠牙髓炎是研究LPS对牙髓组织致病机理的有效、可靠的实验动物模型。②CD14、TLR4参与牙髓炎症的发生发展过程，并在其中发挥作用。③LPS上调CD14、TLR4的表达。更多还原

【Abstract】 Objective: In order to observe and semiquantitative analysis the expression of CD14, TLR4 at different periods and regions in rats’ dental pulp, we established animal model of pulpitis with rats induced by lipopolysaccharide and used immunohistochemistry methods to investigate the role of CD14, TLR4 in pulpitis and provide the experimental evidence of animal for elucidating the pathogenesis of pulpitis which was caused by LPS.Methods: 56 female Sprague-Dawley (SD) rats, 24 weeks old, were randomly divided into 7groups of 8 rats each: one group was normal, other were pulpitis. In groups of pulpitis, First molars in upper jaw of each rat were divided into experimental and control groups. When pulpal tissues were exposured, experimental sides were dealt with LPS as control sides were dealt with NS. The rate were sacrificed at 1, 3, 7days, 2, 3, 4weeks by perfusing 4% paraformaldehyde into heart. The first molars combined with some jaw bones of each rat were fixed, decalcified, embedded in paraffin, and sectioned serially. The sections were stained with hematoxylin-eosin to observe the histopathological changes of pulps. Immunohistochemical staining was used to observe the expression of CD 14 and TLR4. Image pro plus 5 image analysis software was used to determine the average gray value of CD14 and TLR4 positive cells in rat pulpal tissues. Statistical tests were performed by one-way analysis of variance (ONE-way ANOVA), SNK-q test and t test.Results:1. The pulpal tissues survived long more than four weeks in LPS experimental groups. The whole process of the pulpal tissues hurt by LPS and their immunity defence and self-reparation was observed.2. Positive staining for CD14 was not found in any other layer cells of healthy pulp, except some cells in the odontoblast layer. Strong positive staining for CD14 was observed in inflammatory areas (mainly for monocytes/macrophages and neutrophils). Image analysis of CD14 indicated that the difference between normal group and each inflammatory group dealt with LPS had statistical meaning; there were statistical difference among the inflammatory groups, except the difference between three-week group and four-week group; compared LPS-group with NS-group, there were statistical difference in three-day group and seven-day group, no statistical difference in one-day group.3. Positive staining for TLR4 was mainly found in vascular endothelial cells, odontoblasts and inflammatory cells. Statistical results of image analysis of TLR4 was indicated as following: there were statistical difference between normal group and other inflammatory groups deal with LPS except two-week inflammatory group; compared each inflammatory group deal with LPS, except the difference between one-day group and three-day group, other groups shown the statistical difference; compared LPS-group with NS-group, there were statistical difference in every time-group and average gray value of LPS-group was lower than NS-group’s in the same period.Conclusions:1. Experimental model of rats’ pulpitis induced by lipopolysaccharide was the valid and credible animal model for studying the machanism of LPS hurting pulpal tissues.2. It was indicated that CD14 and TLR4 participated in the process of dental pulpitis and played an important role in the pulpitis. 3. LPS can up-regulate the expression of CD14 and TLR4.更多还原