| 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.
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