| Background Papillon-Lefèvre syndrome(PLS) is a rare autosomal recessive disorder, which is characterized by severe early-onset periodontitis. People may suffer this disease from the deciduous dentition and inflammation can continue to permanent dentition with teeth lost finally. Most patients can be accompanied by manifestations of abnormal keratosis of palmoplanter skin. Studies have confirmed that the cathepsin C gene(CTSC) mutation is the pathogenesis of PLS, but the the detailed pathogenic mechanism remains unclear.Cathepsin C, also called Dipeptidyl peptidase I(DPPI), is a kind of lysosomal cysteine protease and is involved in the degradation of proteins in organisms. Evidence has been showed that cathepsin C plays an important part in activating granzyme A and B in cytotoxic T lymphocyte(CTL), thus CTL can take part in normal immune response. Once CTST mutates, the abnormal expression of cathepsin C may weaken immune ability of organisms and induce infection and severe inflammation. However, the reason why the main manifestations of PLS are almost disorders of periodontium and abnormal keratosis of multi-sites cannot be explained clearly. To conclude, exploring and studying the effects of cathepsin C on periodontal inflammation can account for the detailed pathogenesis of PLS in the future.Objective 1. To observe different expressions of cathepsin C in gingival epithelium under different periodontal conditions, and to make a preliminary study on the relationship between cathepsin C expression and the differentiation and immunity condition of gingival epithlium; To clarify the important role which cathepsin C may play in periodontal inflammation. 2. To investigate the effects of CTSC knockout on periodontal inflammation in type C57 mice.Methods 1. To study the different expression of CTSC and cathepsin C in different periodontal conditions: Real-time PCR is used to compare the gene expression of CTSC in healthy group, gingivitis group and periodontitis group; Immunohistochemistry staining is used to compare the protein expression of cathespin C in above groups. 2. To study the relationship between cathepsin C expression and epithelial differentiation in gingival epithelium: We use immunohistochemistry staining to analyse the expression of epithelial differentiation indicator— CK10 and CK19 in healthy group, gingivitis group and periodontitis group. Furthermore, the relationship between CK expression and cathepsin C expression is analysed. 3. To study the relationship between cathepsin C expression and epithelial immunity: We use immunohistochemistry staining to analyse the expression of Toll like receptor 4(TLR4) in healthy group, gingivitis group and periodontitis group. Furthermore, the relationship between TLR4 expression and cathepsin C expression is analysed. 4. To establish the periodontitis model of mice and to evaluate the achievements: We choose the wild type C57 mice and CTSC knockout C57 mice to establish the incisor periodontitis model and molar periodontitis model respectively by injecting Escherichia coli(E.coli) Lipopolysaccharides(LPS) to selected sites of periodontium. After establishing the periodontitis model, we perform a micro-CT scan to the experimental mice and analyse the variation of alveolar bone in the experimental sites. We compare the variation of alveolar bone volume or height of incisors or molars between the wild type C57 mice and CTSC knockout C57 mice; After putting the mice to death, we collecte the periodontal tissues of all experimental mice and make histologic sections for HE staining in order to observe and analyse the inflammation response in different mice. What’s more, immunohistochemical staining of cathepsin C is performed to compare the cathepsin C expression in gingival epithelium of both healthy wild type mice and periodontitis wild type mice. Then further comparation of cathepsin C expression in gingival epithelium in periodontitis is made between human without CTSC mutation and wild type mice.Results 1. Real-time PCR shows that CTSC gene expression in healthy group is much lower than that in gingivitis group and periodontitis group, while the difference of CTSC gene expression in gingivitis group and periodontitis group is not significant. Immunohistochemical staining shows that cathepsin C is scatteredly expressed in healthy gingival epithelium, while in gingivitis and periodontitis gingival tissues, cathepsin C is significantly expressed in all suprabasal cell layers, especially in the surface of epithelium and there is few cathepsin C positive cells in the connective tissues. When we calculate the positive cell ratio of each group, it is found that the positive cell ratio is much higher in gingivitis group and periodontitis group than that in healthy group and the difference of positive cell ratio between gingivitis group and periodontitis group is not significant as well. 2. Immunohistochemical staining shows under periodontal inflammation, the expression of CK10 is strengthened obviously and the expression of CK19 in the suprabasal layers appears. What’s more, the expressions of cathepsin C and CK10 in inflammatory gingival epithelium share a common expression pattern, which features the greatly strengthened expression in suprabasal cell layers, especially in the surface of epithelium. It is implied that cathepsin C might be involved in epithelial differentiation. 3. Immunohistochemical staining shows that under periodontal inflammation, the expression TLR4 is strengthened obviously in almost all cell layers in gingival epithelium, especially in the upper cell layers. In addition, the expression of cathepsin C and TLR4 in inflammatory gingival epithelium nearly share a common expression pattern, which also features the greatly strengthened expression in suprabasal cell layers, especially in the surface of epithelium. It is implied that cathepsin C might be involved in epithelial immune response. 4. By comparing the volume variation of alveolar bone in incisors and height variation of alveolar bone in molars, we find that after establishing the periodontitis models, no matter whether incisor periodontitis model or molar periodontitis model, the variations of bone volume and bone height are quite obvious in wild type mice, while in CTSC knockout mice it is not. Maybe the destruction effects of E.coli LPS on the periodontium is interposed by the CTSC gene mutation. HE staining and immunohistochemical staining show that a lot of inflammatory cells which are positively expressed NF-κB infiltrate in the periodontium of wild type mice while CTSC knockout mice are not and the destruction extent of periodontium is much severer in wild type mice than that in CTSC knockout mice. The results above imply that cathepsin C plays an important part in periodontal inflammation in mice and the absence of cathepsin C may “protect†the periodontium from suffering destruction under the stimulation of E.coli LPS.After comparing the expression of cathepsin C in healthy gingiva and periodontitis gingiva of wild type mice, we find that under healthy condition cathepsin C is mainly expressed in basal cell layer and a few suprabasal cell layers and under periodontitis condition cathepsin C is almost expressed in the whole epithelium except for the surface layer. The expression pattern of cathepsin C in inflammatory gingiva in mice is nearly opposite to that in human, so we infer that cathepsin C has different effects on the inflammation response in periodontium between mice and human and this difference might be induced by species variation.Conclusion 1. The expression of cathepsin C is significantly enhanced in inflammatory gingival epithelium, suggesting that it plays an important role in the inflammation response in periodontium. 2. The variation of cathepsin C expression in gingival epithelium is accompanied by the variation of differentiation and immunity of gingival epithelium. 3. The absence of CTSC gene might has a “protection†effect on the destruction of periodontal tissues induced by inflammatory stimulants, which is absolutely different from the reaction of human beings and this diversity may result from the different expression pattern of cathepsin C and species variation between humans and mice. |
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