SPARC与SHH/PTCH1通路协同调节骨代谢的分子机制研究

Nevoid basal cell carcinoma syndrome (NBCCS), a rare autosomal dominant disease, characterized by multiple skeletal abnormalities, and a propensity for various neoplasms, PTCH1 is the major causative gene. Bone adhesion protein (SPARC / ON / BM-40), an extracellular matrix protein, is thought to be involved in bone metabolism mainly by binding to Ca2+ and collagen. Our previous study found that in non syndromicodontogenickeratocyst (PTCH1+/+) mesenchymal cells as control, syndrome type of odontogenickeratocyst (PTCH1+/-) mesenchymal cells showed weaker osteogenic differentiation ability; and protein mass spectrometry showed that SPARC lower expression in S-OKC mesenchymal cells, especially with PTCH1 protein truncation mutations were significantly down regulated. PTCH1 gene knockdown downregulated SPARC expression in MG63 cells. Indicated that Shh-PTCH1 signaling pathway may be involved in the regulation of SPARC on bone metabolism. In this research, we will analysis the correlation between types of PTCH1 mutation that NBCCS patients carried and serum SPARC expression; Then we construct Shh-PTCH1 signaling pathway activating/inhibition/silencing cell models, evaluate the expression and localization of SPARC and cells osteogenic differentiation; Moreover we apply graded concentrations of SPARC for osteogenic differentiation, meanwhile blocking Smad/MAPK pathway, detect the status of calcium channel opening and osteogenic differentiation. In this study, we intensivly explore the molecular mechanisms involved in the regulation of Shh-PTCH1 signaling pathway and SPARC expression, definite the role and mechanism of SPARC in osteogenic differentiation. And will provide a theoretical basis for the monitoring of bone metabolism in patients with NBCCS levels, and find the treatment target, adverse effects on systemic reverse gene abnormal bone metabolism.

痣样基底细胞癌综合征是一种罕见的常染色体显性遗传病，PTCH1为主要致病基因，常表现骨骼的发育异常，然而其骨骼异常的机制尚不明确。我们前期研究发现，综合征型角化囊肿（PTCH1+/-）间充质细胞的成骨分化能力弱，伴随患者全身骨密度降低，且SPARC在该间充质细胞中（尤其携带PTCH1蛋白截断突变者）显著下调。我们假设，Shh/PTCH1/SPARC可调节骨代谢平衡。我们将对综合征患者PTCH1突变类型与血清SPARC的表达进行相关性分析；分别构建Shh－PTCH1通路激活/抑制/沉默细胞系，评价SPARC的表达、定位、细胞成骨分化；用不同浓度SPARC诱导细胞成骨分化，及阻断Smad/MAPK通路后，检测钙离子通道开放及成骨分化。本研究将深入阐述Shh-PTCH1通路调控SPARC表达的机制，明确SPARC在成骨分化过程中的调节机制，为监测NBCCS患者骨代谢水平，寻找治疗靶点提供依据。

痣样基底细胞癌综合征（NBCCS），是一种罕见的常染色体显性遗传病，常表现骨骼的发育异常，PTCH1为主要致病基因。骨粘连蛋白（SPARC）是一种细胞外基质蛋白,可能通过与Ca2+及胶原结合的方式参与骨代谢。我们前期研究发现，以非综合征型角化囊肿（PTCH1+/+）间充质细胞为对照，综合征型角化囊肿（PTCH1+/-）间充质细胞的成骨分化能力弱，伴随患者全身骨密度降低，且SPARC在PTCH1+/-间充质细胞中（尤其携带PTCH1蛋白截断突变者）表达显著下调，提示Shh－PTCH1信号通路可参与SPARC对骨代谢的调控。在此基础上，我们进一步扩大临床研究样本，探索PTCH1基因突变在NBCCS患者骨代谢中的作用。共收集培养综合征型KCOT间充质细胞21例，PTCH1基因突变检测显示21例综合征型KCOT间充质细胞中有17例携带有PTCH1基因突变，突变类型与外周血一致。骨密度检测结果显示NBCCS患者骨密度值低于正常对照人群，且携带PTCH1基因截断突变类型的NBCCS患者骨密度值降低更明显，与PTCH1基因非截断突变型NBCCS患者骨密度值具有统计学差异。通过破骨细胞的诱导实验，综合征型间充质细胞诱导破骨代谢的活性明显高于非综合征型。同时，我们通过组织学及细胞学检测SPARC的表达及定位情况，发现综合征型间充质细胞SPARC表达下调，与PTCH1突变一致。因此进一步探索SPARC对成骨分化的促进作用及其机制，将不同浓度的SPARC作用于人骨髓间充质干细胞的成骨诱导过程，用ALP 活性、成骨相关基因（Runx-2、COLI、OCN、OPN、BSP）的表达，以及矿化结节形成量评价细胞成骨分化的情况，发现外源性加入SPARC可促进骨髓间充质干细胞的成骨向分化。.综上，综合征型KCOT间充质细胞比非综合征型KCOT间充质细胞具有更强的增殖活力和克隆形成能力，且负向骨代谢活跃。SPARC表达下调与PTCH1突变导致剂量不足密切相关，进一步影响NBCCS患者骨密度水平。

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