SHH通路抑制剂对牙源性角化囊性瘤病变细胞体外生长干预影响的研究

Keratocystic odontogenic tumor (KCOT) is a common odontogenic lesion, which may occur sporadically or in association with nevoid basal cell carcinoma syndrome (NBCCS). It is locally aggressive, may cause marked destruction of the jaw bones and has a propensity for recurrence. Our previous studies have indicated that frequent PTCH1 mutations (over 80%) were detected in both NBCCS related and sporadic KCOTs, suggesting that PTCH1 misfunction may constitutively activate Sonic Hedgehog (SHH) signaling and could play a major role in the pathogenesis of KCOT. Small molecule drugs targeted to inhibit SHH signaling may provide new treatment strategies for KCOT. This project was to construct in vitro cellular models for PTCH1 misfunction/inactivation by introducing PTCH1 mutations of KCOT patients into human embryonic stem cell line (hESC) using CRISPR/Cas9 gene editing technique. Through inductive epithelial and fibroblastic differentiation of the hESC, the status of SHH signaling and biologic behavior of the lesion cell models was investigated in vitro. Small molecule SHH inhibitors were then screened and tested for their effects on the cell proliferation and apoptosis as well as the osteogenic and osteoclastogenic activities in the induced hESC and KCOT lesion cells. Thus this study will provide further insight into the molecular mechanism of KCOT and new strategies for KCOT treatment of molecular approaches.

牙源性角化囊性瘤（KCOT）是一种颌骨牙源性病损，可单发或多发，后者常伴发痣样基底细胞癌综合征（NBCCS）。KCOT生长具有局部侵袭性，可导致颌骨严重破坏，术后易复发。我们前期研究发现：NBCCS相关性及散发性KCOT均频发PTCH1基因突变（>80%），提示PTCH1失活导致Sonic Hedgehog（SHH）信号通路的组成性激活，可能是KCOT发生的重要机制。靶向抑制SHH信号通路的小分子药物可能为KCOT的治疗提供新方法。本课题拟利用CRISPR/Cas9基因编辑技术，将KCOT患者PTCH1突变引入人胚胎干细胞（hESC），在体外构建PTCH1基因失活突变的细胞模型，并向上皮和成纤维细胞诱导分化，体外检测诱变细胞SHH通路的激活状态和生物学特点，同时筛选、观察SHH抑制剂对诱变hESC和KCOT病变细胞增殖、凋亡、成骨、破骨能力的影响，为探索分子靶向治疗方法提供实验依据。

背景：.牙源性角化囊性瘤（keratocystic odontogenic tumor，KCOT）是常见的颌骨囊性病损，其生长具有局部侵袭性，可散发，亦可伴发发痣样基底细胞癌综合征（nevoid basal cell carcinoma syndrome，NBCCS）。前期研究发现：NBCCS相关性及散发性KCOT的上皮衬里均频发PTCH1基因突变, 提示PTCH1功能失活性突变，导致非配体依赖型SHH信号通路的组成性激活，可能是KCOT发生的重要分子机制，靶向抑制SHH信号通路的小分子药物可能为KCOT的治疗提供新方法。但由于KCOT细胞模型及动物模型的缺乏，目前除了零星个案报告外，尚缺乏SHH通路小分子抑制剂治疗KCOT的相关机制研究。.研究内容：.本研究利用CRISPR/Cas9基因编辑技术，将KCOT患者PTCH1杂合突变c.403C>T (p. R135X) 引入到人胚胎干细胞（human embryonic stem cell，hESC），体外构建PTCH1杂合突变（PTCH1R135X /+）hESC细胞模型，并向上皮细胞方向诱导分化，获得模拟KCOT的病变上皮细胞模型PTCH1R135X /+ hESC-E。利用该模型检测诱变细胞SHH通路的激活状态和体外增殖特点，同时观察SHH通路小分子抑制剂GDC-0449对诱变细胞SHH通路、体外增殖的影响。.结果：.CRISPR/Cas9定点编辑及抗生素筛选后获得耐药克隆30个，综合突变位点PCR、5’ junction PCR、3’ junction PCR、Western Blot结果以及细胞状态，选择PTCH1R135X/+杂合突变模型43#-hESC向上皮前体细胞诱导分化，获得模拟KCOT病变上皮细胞模型43#-hESC-E。利用该模型，体外验证了PTCH1基因单倍体剂量不足可导致SHH通路非配体依赖型激活、细胞增殖能力增强；用合适浓度的GDC-0449阻断其异常激活的SHH通路可抑制其变强的增殖能力，提示GDC-0449可成为靶向治疗KCOT的有效药物。.结论：.本研究对解决供体细胞来源、研究KCOT发病机制及分子靶向治疗进行了初步探索，所建立的PTCH1诱变细胞模型PTCH1R135X/+ hESC-Es为KCOT相关研究提供了新手段。