纤维粘连蛋白基因可变剪接对牙源性囊肿样病变颌骨组织破坏的调控

The odontogenic cystic lesions, including series of frequent maxillofacial disease, always lead to local bone destruction. Recently, the association between the fibrous wall and bone resorption became the focus of many scholars..In our previous studies, the stroma of the fibrous wall surrounding the keratocystic odntogenic tumor (KCOT) induced osteoclastogenesis effectively, it could be attributed to the direct cell to cell interation between osteoclasts precursor and fibroblasts; or indirectly to the changed paracrine as a result of the interaction between fibroblasts and epithelia. It suggested the important role of the stroma in the bone destruction of odontogenic cystic lesions. Further the changes of the component of extracellular matrix (ECM), such as increased LOXL4 contributed the progression of KCOT via angiogenesis, suggesting the microenvironment composed of various ECM components promotes the progression or local aggressiveness of the odontogenic cystic lesions, as well as the bone destruction..As an important component of ECM, fibronectin (FN) could be investigated in various odontogenic cyst, in which the alternative splice of FN gene may be involved in the osteoclasts differentiation, and hence the local bone destruction, since the FN isoforms generated by alternative splice were always involved in osteoclastogenesis via various cytokines, as investigated in various diseases. According to our recent studies, the mRNA of the isoform EDA+FN positively associated with the range of cysts, therefore the FN gene is likely to participate in the local destruction of the jaw bone. Correspondingly, the changes of the ratio of the FN isoforms in the ECM may be a strategy to inhibited the lesion progression, as supported by our previous study, in which the exon of EDA was knockout from FN gene, and hence the aggressiveness, motility as well as proliferation of salivary adenoidcystic carcinoma (SACC) were significantly down-regulated..Through this project, we propose investigating and explicating the derivation, types and distribution of the FN isoforms in the odontogenic cystic lesions, as well as the their associations with osteoclastogenesis. It is helpful to understand the mechanism of ECM participating in the jaw bone destruction. On this basis, to change the alternative splice of FN by CRISPR/Cas system may provide a strategy, by which the modified microenvironment of ECM could inhibit the progression of not only odontogenic cystic lesions, but also other pathological condition.

牙源性囊肿样病变，包括一系列破坏颌骨的口腔颌面部疾病。近年来其间质与骨组织破坏的关系逐步受到重视。前期工作证实，通过改变细胞外基质（extracellular matrix，ECM）组分，或与上皮或破骨细胞前体相互作用，病变间质直接或间接介导破骨细胞发生。ECM中纤维粘连蛋白（Fibronectin，FN）经可变剪接产生各种异构体，分布于不同病变并参与骨破坏。本项目组还发现，牙源性角化囊性瘤（keratocystic odontogenic tumor, KCOT）囊壁成纤维细胞中，FN基因可变剪接外显子EDA的mRNA水平与颌骨破坏范围正相关，提示FN异构体对颌骨破坏过程至关重要。本项目拟探索牙源性囊肿样病变中FN异构体种类、来源和分布与局部骨破坏的关系及相应分子机制；在前期基础上利用CRISPR/Cas9编辑FN基因，改变ECM成分及微环境，控制病变骨破坏。

本项目采用根尖周囊肿为疾病模型，致力于探讨成纤维细胞与骨组织破坏的关系。根尖周囊肿囊壁内，包含EDA外显子的纤维粘连蛋白（Fibronectin, FN），EDA+FN表达程度与囊肿的病损面积及囊壁内CD34阳性血管管腔面积成显著正相关；而体外培养的成纤维细胞中，EDA+FN的相对表达量与成纤维细胞诱导形成的破骨细胞数量显著正相关。EDA+FN主要通过自分泌作用于成纤维细胞本身，刺激后者上调一系列破骨分化相关基因表达，从而刺激破骨细胞形成；其中血管内皮生长因子（Vascular endothelial factor, VEGF）是直接促进破骨细胞形成的主要分子。进一步从成纤维细胞中分离克隆形成单位（Colony forming units，CFUs）发现，属于第二类亚群的成纤维细胞是诱导破骨细胞形成的主要亚群，该亚群正是依赖于EDA+FN刺激VEGF的细胞。通过体外基因编辑成纤维细胞，敲除其EDA外显子后注入动物骨缺损模型，可以显著降低体内破骨细胞形成效率。