PTH诱导血管内皮-软骨细胞转分化在CKD血管钙化发生中的作用和机制研究

Vascular calcification (VC) is a prominent feature of vascular remodeling in patients with chronic kidney disease (CKD) and is a critical risk factor for increased cardiovascular morbidity and mortality. Studies have indicated that serum parathyroid hormone (PTH) is increasing with the decline of renal function and has been demonstrated to associate with the development of VC. However, the underlying mechanisms are still unclear. Our previous work has suggested that endothelial-to-mesenchymal transition (EndMT) contributes to the accumulation of chondrocyte during the media calcification process. In addition, PTH could directly induce EndMT and acquisition of mesenchymal stem cell-like phenotype in cultured aortic endothelial cells. This study will be performed based on our novel scientific hypothesis that PTH as a critical element which stimulate the formation of VC in CKD by inducing phenotypic change of endothelium to chondrocyte. To address this, primary endothelial cells culture and multiple animal models of PTH-associated VC will be used to explore the effect of PTH on endothelial-to-chondrocyte transition and the involvement of Wnt/β-catenin signaling pathway by the ways of cell-lineage tracing,transgenic, molecular imaging, cellular and molecular biology technologies. This study will lead to the clarification of potential mechanisms of VC in CKD patients and finally benefit the preventive strategy and treatment of VC with CKD.

血管钙化（Vascular calcification, VC）是慢性肾脏病（CKD）患者病程中重要的血管重塑，是导致心血管疾病发病率和死亡率显著增加的危险因素。CKD患者随着肾功能下降而引起的血液中甲状旁腺激素（PTH）水平升高与VC形成密切相关，但其确切机制尚未阐明。本课题组前期发现，内皮细胞通过内皮间充质转分化（EndMT）成为软骨细胞可能参与血管中膜钙化的形成。PTH可以诱导主动脉内皮细胞发生EndMT，并表达间充质干细胞标志物。本研究拟在前期工作基础上，利用细胞谱系追踪、基因敲除等模式动物和体外细胞培养技术，采用病理形态学、影像学和细胞及分子生物学等分析方法，探讨PTH诱导内皮－软骨细胞转分化在血管钙化形成中的作用及与Wnt/β-catenin信号通路的关系，以阐明PTH在促进CKD血管钙化形成过程中的作用和机制，为早期防治CKD血管钙化提供理论依据。

高PTH血症可诱导血管钙化病变的形成，但其具体机制仍不十分清楚。近年来，研究证实内皮细胞发生间充质转分化（EndMT）后可参与异位钙化病变的形成。因此，本研究拟通过体内外实验观察EndMT是否参与介导高PTH血症相关CKD血管钙化的形成，并初步探讨上述细胞转化过程的潜在分子机制。. 首先，我们在慢性肾功能衰竭（CRF）模型大鼠中观察到： CRF组大鼠主动脉中膜钙化面积较对照组显著增加，并伴随EndMT相关标志物和软骨细胞标志蛋白的表达增加；而接受CINA降PTH治疗的CRF模型大鼠主动脉中膜钙化病变则较CRF模型组大鼠显著减轻，且EndMT相关标志物和软骨细胞标志蛋白的表达均下调。继而，我们给予内皮细胞荧光示踪小鼠超生理剂量的PTH灌注后发现，模型组小鼠主动脉壁内出现内皮细胞和软骨细胞标志物的共表达。随后，体外实验结果显示，超生理剂量PTH能够诱导人主动脉内皮细胞（HAECs）EndMT相关标志物的表达上调；后者在接受软骨培养基培养后，软骨细胞标志物和细胞钙含量均显著增加。同时，PTH可诱导HAECs发生β-catenin核转位；应用β-catenin siRNA或DKK1干预可部分阻断PTH所诱导的EndMT相关标志物的表达，在软骨细胞诱导培养后软骨标志蛋白水平也显著下降。随后，在基础实验的基础上，我们进一步进行了钙化防御（calciphylaxis）的临床观察性研究，结果证实钙化防御患者的影像学检查显示全身多发异常钙化灶和骨质改变，对该类患者进行皮肤活检病理学检查有助于早期诊断。. 此外，近年来，基础和临床研究发现CKD血管钙化是与骨代谢异常密切相关的的生物学过程。因此，我们在血管钙化发病机制的研究基础上探讨骨质疏松的发生机制。研究结果显示：对照组大鼠股骨松质骨结构完整，而CRF组存在明显的松质骨减少，结构紊乱；CKD组腰椎骨和股骨骨密度，最大应力及刚度均较对照组减少。进一步应用PTH干预体外培养的HAECs，发现PTH能够诱导内皮细胞发生脂肪细胞转分化。上述研究共同提示了，CKD患者骨质疏松的发生机制可能与脂肪细胞浸润增加有关。