【作者】 徐莉；

【导师】 许丽华；

【作者基本信息】 河北医科大学 ， 口腔临床医学， 2010， 硕士

【摘要】 目的:研究大鼠β-防御素-1,2(β-defensin-1, 2, RBD-1, RBD-2)在牙周炎模型牙龈上皮及腮腺导管上皮的分布,探讨RBD-1、RBD-2在大鼠牙周炎发生、发展过程中的作用,从而为阐明防御素在牙周免疫防御中的作用提供初步的理论依据。方法:选用纯种6月龄雄性Wistar大鼠30只,随机分为三组:正常组、牙龈炎组、牙周炎组,每组各10只。牙龈炎和牙周炎组用直径0.2mm的正畸结扎丝结扎大鼠双侧上颌第一磨牙,并置于龈下。随机分为两组:结扎2周的为牙龈炎组,结扎6周的为牙周炎组。牙龈炎组大鼠于试验的第2周,牙周炎组和正常组大鼠于试验的第6周分别股动脉放血处死。取大鼠上颌骨连同牙及牙周组织、腮腺及其部分导管分别置于10%甲醛缓冲液固定48h,10%乙二胺四乙酸(EDTA)脱钙40d。乙醇梯度浓度脱水,石蜡包埋。将各组标本制成颊舌向4μm厚的组织切片,用SP免疫组化染色法分别检测观察三组大鼠牙周、腮腺及其导管中RBD-1、RBD-2蛋白表达情况。结果:1牙龈炎和牙周炎动物模型的建立1.1肉眼观察:大鼠结扎2周后,肉眼观察大鼠上颌第一磨牙牙龈呈暗红色,龈乳头和龈缘红肿,探诊出血,但未探及明显的牙周袋,故诊断为牙龈炎;当大鼠结扎6周后,除牙龈红肿,探之出血加重外,还可探及深牙周袋,故诊断为牙周炎。1.2 HE染色结果:1.2.1牙周组织的HE染色结果正常组:沟内上皮完整,有角化,角化程度较口腔龈上皮差,结合上皮位置正常,牙龈上皮无明显炎症细胞浸润,牙龈及牙周膜纤维排列有序、规律,牙槽骨边缘整齐,未见破骨细胞。牙龈炎组:沟内上皮完整,结合上皮位置正常,牙龈的纤维结缔组织水肿明显,其间有炎症细胞浸润,毛细血管数目增多、管腔扩张充血。上皮钉突数目增多,伸长,牙周膜纤维排列正常,牙槽骨边缘整齐,未见破骨细胞。牙周炎组:沟内上皮溃疡、坏死,结合上皮向根方增殖,上皮和固有层内有大量炎症细胞浸润,牙周膜纤维排列紊乱,毛细血管数目增多、管腔扩张充血,牙槽骨内可见破骨细胞。1.2.2腮腺的HE染色结果:正常组:管壁上皮细胞排列整齐,胞核圆形且较大,管腔内无炎症细胞浸润,导管周围及腺体内亦无炎症细胞浸润。牙龈炎组与牙周炎组腮腺组织表现与正常组相似。2免疫组化观察2.1牙龈组织中RBD-1、RBD-2的表达情况在正常组、牙龈炎组和牙周炎组口腔龈上皮和沟内上皮均可观察到RBD-1表达的棕黄色阳性染色颗粒,即RBD-1在三组中均有表达,阳性染色区域主要分布在上皮细胞的胞浆内。包括角化层、颗粒层、棘层,主要分布于棘层近颗粒层的细胞胞浆内,强染色区域呈带状或片状分布,基底细胞未见明显着色。与之相比,沟内上皮着色较浅。但在结合上皮、上皮下结缔组织及骨组织中未见到RBD-1表达。RBD-2的表达情况与RBD-1的表达情况相似,均只在上皮部位表达,结缔组织及骨组织未见表达。2.2腮腺导管中RBD-1、RBD-2的表达情况正常组、牙龈炎组和牙周炎组腮腺导管中均可观察到RBD-1表达的棕黄色阳性染色颗粒,即RBD-1均有表达,阳性染色区域主要分布在腮腺导管的上皮细胞的胞浆内,腮腺腺泡不着色。RBD-2的表达情况与RBD-1的表达情况相似。3半定量分析结果RBD-1在口腔龈上皮的阳性细胞计数在正常组、牙龈炎组和牙周炎组无明显差异(P>0.05)。在沟内上皮中,RBD-1在正常牙龈组的阳性细胞计数显著高于牙龈炎组和牙周炎组(P<0.05),而牙龈炎组和牙周炎组之间无显著性差异(P>0.05);三组中RBD-1在口腔龈上皮中的表达均明显高于沟内上皮(P<0.05)。RBD-2在口腔龈上皮的阳性细胞计数在牙龈炎组高于正常组和牙周炎组(P<0.05),正常组高于牙周炎组(P<0.05);在沟内上皮中,RBD-2在正常组的阳性细胞计数显著高于牙龈炎组和牙周炎组(P<0.05),而牙龈炎组和牙周炎组之间无显著性差异(P>0.05);三组中RBD-2在口腔龈上皮中的表达均明显高于沟内上皮(P<0.05)。RBD-1在腮腺导管上皮的阳性细胞计数在正常组、牙龈炎组和牙周炎组无明显差异(P>0.05)。RBD-2在腮腺导管上皮的阳性细胞计数在正常组、牙龈炎组和牙周炎组亦无明显差异(P>0.05)。结论:1.成功建立了大鼠牙龈炎、牙周炎的动物模型。2. RBD-1、RBD-2在正常及牙龈炎、牙周炎状态下的牙龈组织中都有表达,主要表达于口腔龈上皮,在沟内上皮有微弱表达,结合上皮、结缔组织及骨组织均无表达。3. RBD-1在正常及牙龈炎、牙周炎状态下的口腔龈上皮组织中都有表达,其表达强度无明显差异;RBD-2在正常及牙龈炎、牙周炎状态下的口腔龈上皮组织中亦都有表达,但在牙龈炎状态下表达强度明显升高,在牙周炎状态下表达强度显著降低。4. RBD-1、RBD-2在正常和牙龈炎、牙周炎状态下的腮腺组织中都有表达,主要位于腮腺导管上皮细胞,各组间无明显差别。更多还原

【Abstract】 Objective: To Study the distribution of RBD-1, RBD-2 in the gingival epithelial cells and the parotid duct of rat periodontitis and explore the role of RBD-1, 2 in the occurrence and development of rat Periodontitis, so to provide basic data for further exploring the possible role of rat denfensins in the periodontal immune defense.Methods: Thirty male Wistar rats were radomly divided into three groups: normal group(group normal), gingivitis group(group G), and periodontitis group(group PD), 10 rats in each group. Twenty rats were ligated of their bilateral maxillary first molar teeth with 0.2mm steel-wire being placed in subgingival. They were randomly divided into two groups. The rats which were ligated two weeks were group G, and the rats which were ligated six weeks were group PD. The rats in group G, group PD and group normal were respectively bled to death by femoral arteries bleeding at 2th, 6th, 6th week. The rat maxilla together with the surrounding periodontal tissues, parotid gland and part of its duct were collected and placed in 10% formaldehyde 48 hours, and was put into 10%EDTA for decalcification for 40days, and embedded in paraffin. The embedded samples were routinely sectioned to yiled 4μm thick slices. The expression of RBD-1 and RBD-2 protein in the gingival tissues and parotid ducts in the three groups was detect by SP immunohistochemical staning method.Results:1 Animal model of Simple gingivitis and periodontitis was established successfully1.1 Clinical observationAt the 2th week after ligation, the gingival around the first molar teeth became dark red by visual observation. It’s edema in the gingival nipples and gingival margin, bleeding after probing, but revealed no significant periodontal pocket, so it was diagnosed as gingivitis; At the 6th week after ligation, the above symptoms at two weeks became more obvious, and periodontal pocket could be detected. so it was diagnosed as periodontitis.1.2 HE staining results1.2.1 The HE staining results of periodontal tissueGroup normal: The sulcus epithelium was integrity and has keratosis, but, thinner than keratinized oral gingival epithelium. The position of the junctional epithelium was normal, and there was no inflammatory cell in the gingival epithelium. Gingival and periodontal ligament fibers were arranged orderly and regularly. The edge of alveolar bone was neat and there was no osteoclasts.Group G: The sulcus epithelium was integrity. The position of the junctional epithelium was normal. It’s edema significantly in gingival fibrous connective tissues. There was inflammatory cell infiltration, increased number of capillaries, lumen dilatation and congestion. Epithelial nail increased in number. Gingival and periodontal ligament fibers were arranged orderly and regularly. The edge of alveolar bone was neat and there was no osteoclasts.Group PD: There were ulcers and necrosis in sulcus epithelium. The junctional epithelium proliferated toward the root side. There was moderate inflammatory cell in the epithelium and lamina propria. Periodontal ligament fibers were disordered and the number of capillary were increased. There were osteoclasts in alveolar bone.1.2.2 The HE staining results of parotid glandGroup normal: Wall epithelial cells arranged in neat rows, round nucleus and a larger lumen without infiltration of inflammatory cells. Around the duct and the gland there was no inflammatory cell infiltration.The HE staining results of parotid gland in group G and group PD were similar to the normal group.2 Immunohistochemical Observation2.1 The expression RBD-1, RBD-2 in gingival tissues The brown positive staining granules of RBD-1 could be observed in oral gingival epithelium and sulcus epithelium of the group normal, the group G and the group PD. The positive staining areas are mainly distributed in the cytoplasm of epithelial cells, including the keratinizing layer, granular layer, acanthosis, mainly distributed in the spine-layer near the cytoplasm of granular layer cells, strong staining formed a positive band distribution pattern, basal cell revealed no obvious expression. In contrast, the sulcular epithelium showed only weak expression. However, in the context of epithelial cells and the sulcular epithelium connective tissue and bone tissue did not see the RBD-1 expression.The expression pattern of RBD-2 was similarwith that of RBD-1.2.2 The expression RBD-1, RBD-2 in the parotid ductThe brown positive staining granules of RBD-1 could be observed in the parotid duct of the group normal, the group G and the group PD. The positive staining areas are mainly distributed in the cytoplasm of the parotid gland duct epithelial cells.There was no expression within the parotid gland cellsThe expression pattern of RBD-2 was similarwith that of RBD-1. 3 Semi-quantitative analysis of RBD-1,2 immunohistochemistry staningThe positive cell number of the RBD-1 in oral gingival epithelium showed no significant difference in the group normal, group G and group PD (P>0.05); In the sulcular epithelium, the positive cell number of the RBD-1 protein significantly higher in group normal compared with group G and group PD(P<0.05), while there was no significant difference between the group G and group PD(P>0.05); The expression of RBD-1 was significantly higher in the oral gingival epithelium than in the sulcular epithelium(P<0.05).The positive cell number of the RBD-2 significantly higher in group G compared with group normal and group PD(P<0.05); in the sulcular epithelium, the positive cell number of the RBD-2 protein significantly higher in group normal compared with group G and group PD(P<0.05), while there was no significant difference between the group G and group PD(P>0.05); The expression of RBD-2 was significantly higher in the oral gingival epithelium than in the sulcular epithelium(P<0.05).The positive cell number of the RBD-1 in the parotid duct epithelial showed no significant difference in the group normal, group G and group PD (P>0.05).The positive cell number of the RBD-2 in the parotid duct epithelial showed no significant difference in the group normal, group G and group PD (P>0.05).Conclusion: 1. The animal model of gingivitis and periodontitis were successfully established by the first molar ligation method. 2. The expression of RBD-1, RBD-2 could be found in the gingival tissues of normal, gingivitis and periodontitis, primarily expressed in the oral gingival epithelium, weakly expressed in sulcular epithelium, negatively expressed in junctional epithelium, tissue and bone tissue. 3. The expression of RBD-1 could be found in oral gingival epithelial tissues of normal, gingivitis and periodontitis, and showed no Significant difference in the three group; The expression of RBD-2 could be found in oral gingival epithelial tissues of normal, gingivitis and periodontitis, but, significantly higher in group gingivitis, lower in group periodontitis compared with group normal. 4. The expression of RBD-1, RBD-2 could be found in parotid duct in the three group, primarily expressed in the parotid duct epithelial cells, and showed no significant difference in the three groups.更多还原