组织蛋白酶C基因突变导致掌跖角化-牙周破坏综合征的分子机制

Papillon-Lefèvre syndrome (PLS,MIM 245000) is an autosomal recessive disease which is mainly characterized by severe early onset periodontitis and hyperkeratosis involving the palms and soles. Mutations in the gene encoding cathepsin C (CTSC, MIM 602365) have been reported in PLS patients. Loss-of-function mutations in cathepsin C leading to defects in neutrophil serine proteases have been identified in PLS patients. Neutrophils from these patients have minimal residual activities in all three serine proteases. The bactericidal activity of neutrophils depends partly on the presence of these granule-associated proteases and the generation of neutrophil serine protease-deficient mice confirmed the relative importance in the killing of certain Gram-negative and Gram-positive bacteria. Loss of neutrophil serine protease activity may also affect the processing of human 18-kDa antimicrobial protein (hCAP18) to the antimicrobial peptide LL-37 which may also potentially explain the susceptibility of PLS patients to periodontal diseases, but the relationship between palmoplantar hyperkeratosis and CTSC gene mutation is not clear. .The purpose of this study is to investigate the molecular mechanism of PLS and the role of CTSC gene. For functional analysis, our experiment was designed into three groups: wild type gene, mutant gene and gene silence groups. First, the mutations c.778T>C，c.774C>G and c.851G>A were introduced into the wild-type CTSC construct through site-directed mutagenesis, and then the wild type CTSC and mutant type CTSC were constructed in the pEGFP-C1 plasmids. Human keratinocyte line HaCat cells were cultured and gene transfection was carried out using Lipofectamine 2000. We also used RNA interfere technique to silence gene expression in HaCat cells. After transfection and RNAi, real-time quantitative PCR and western blot were performed to analysis the expression of keratin1, keratin9 and loricrin. After transfection and RNAi, cells were fixed and stained with anti-tubulin antibody, and subsequently with anti-keration antibody to detect keratin filament network. Images were taken using a confocal laser scanning microscope.On the basis of preliminary work,our study hope to explain the possible mechinism of CTSC gene in PPK.

掌跖角化-牙周破坏综合征（Papillon-Lefèvre Syndrome，PLS，MIM 245000）一种常染色体隐性遗传性疾病，疾病表现为发生在乳牙列和恒牙列的早发性牙周炎和掌跖皮肤过度角化。CTSC 基因是其致病基因。目前已经证实牙周组织微环境的特殊性和患者存在中性粒细胞的功能缺陷，是导致PLS早发性牙周破坏的原因，但是CTSC基因与患者掌跖皮肤角化之间的联系尚未明确。本研究在前期工作的基础上，构建CTSC基因野生型和突变体的表达质粒，转染人永生化表皮细胞（HaCat）标准株；另外通过RNAi技术进行细胞水平的基因沉默。通过实时定量PCR和western blot技术分析角蛋白和兜甲蛋白在mRNA和蛋白质水平表达的差异，并且通过激光共聚焦显微镜观察Hacat细胞内角蛋白丝形态学的改变，探讨CTSC基因是否通过影响角蛋白或兜甲蛋白导致皮肤角化，初步揭示PLS皮肤角化的分子机制。

掌跖角化-牙周破坏综合征是一种由组织蛋白酶C(CTSC)基因突变引起的罕见遗传疾病，主要表现为掌跖角化和严重的早发性牙周炎。本课题通过研究CTSC对角质形成细胞增殖和凋亡的影响，探讨CTSC缺失与掌跖角化临床表型之间的关系。通过将携带野生型CTSC基因的质粒和CTSC靶向的siRNAs转染HaCaT细胞，研究CTSC的表达对细胞角化的影响。实时定量PCR及蛋白免疫印迹实验（Western Blot， WB）分析表明兜甲蛋白（loricrin，lor）和角蛋白1（cytokeratin 1，KRT1）的表达受CTSC表达的影响，而角蛋白9（cytokeratin 9，KRT9）则不受影响。CTSC过表达时lor表达量升高，CTSC敲低时lor表达量下调，同时发现lor的表达和细胞凋亡现象有正相关关系。当CTSC过表达时，KRT1表达量下调；CTSC敲低则KRT1表达升高，在HaCaT细胞中可观察到明显的点状KRT1聚集颗粒。本研究表明通过敲低CTSC导致lor表达下调而促进细胞增殖是过角化形成的可能机制。