PTHrP/Wnt双信号通路在软骨肉瘤细胞中交互作用机制及去分化调控研究

Chondrosarcoma is the second most common primary bone cancer with poor response to chemotherapy and radiotherapy. There are several different types of chondrosarcoma based on the type of cells identified under a microscope. De-differentiated chondrosarcoma, along with mesenchymal chondrosarcomas, are highly malignant, particularly aggressive. But the mechanism of its de-differentiation regulation is unknown. Parathyroid hormone-related protein (PTHrP) is a key signaling in epiphyseal growth plate development by delay differentiation of chondrocyte and its expression has been demonstrated in a large number of tumour-derived cell lines with progression of tumour malignancy. Wnt/?-catenin signaling plays a vital role in regulating bone formation, metabolize and is applied to osteosarcoma targeted-therapy. Several studies have explored the cross-talk between PTHrP and Wnt signaling pathways.They showed that PTH(1-34) could change the expression of several components of the canonical Wnt pathway and increases stabilized ?-catenin, More recently evidence has shown PTH/PTHrP receptor binding to Wnt co-receptor Lrp6 and stabilization of ?-catenin in osteoblasts. Here We employ pTet-on-off plasmid, ShRNA technic, quantum dot mark technic, et al to perform bidirectional regulation of this two signaling and study how they cross-talk in the chondrosarcoma cells and its effection and mechanism on de-differentiation regulating. It will help us to make a further approach to find a new opportunities that lie within the signaling pathway to be used as targets in oncology drug discovery for chondrosarcoma.

软骨肉瘤是发病率仅次于骨肉瘤的第二大骨组织恶性肿瘤，其分化越差，恶性度越高，对放化疗不敏感，但参与其发生发展的信号调控机制仍不明确。最新研究表明骺板区软骨细胞分化调控异常与软骨肉瘤形成密切相关 ，PTHrP信号是骺板软骨细胞分化调控的关键信号，Wnt信号是骨形成、代谢及骨肉瘤中关键信号，?-catenin是两条通路的关键交汇点。已有证据显示Wnt信号也参与软骨分化调控，而且两者间被证实存在多级交互作用。去分化软骨肉瘤因其含有不同分化级别的软骨细胞，为研究提供良好平台。本项目拟利用可调控基因表达、RNA干扰、量子点标记等技术实现双信号双向调控，通过对信号通路活性及细胞分化、增殖情况的检测，研究PTHrP/Wnt双信号通路在去分化软骨肉瘤细胞系中交互作用特点以及对细胞异常分化、增殖的调控作用及机制，为寻找新的软骨肉瘤治疗靶点提供理论依据。

项目背景：PTHrP信号是骺板软骨细胞分化调控的关键信号，Wnt信号是骨形成、代谢及骨肉瘤中关键信号，β-catenin是两条通路的关键交汇点，两者间被证实存在多级交互作用，共同参与软骨的分化调控，二者间异常表达调控可能导致软骨肉瘤的发生。.主要研究内容：通过研究不同分化级别软骨肉瘤组织标本中PTHrP/Hedghoge信号通路及Wnt信号通路相关蛋白的表达情况及差异。应用重组人PTH和DKK1细胞因子实现对PTHrP/Hedghoge和Wnt通路的双向调控，明确二者相关的primary cilia在调控软骨肉瘤去分化过程中的作用。 通过可调控的信号通路的活化或者抑制，明确二者的交互作用在软骨肉瘤细胞去分化过程中的作用机制，并探讨primary cilia是否是影响软骨肉瘤细胞去分化的关键作用位点。.重要结果：我们发现不同分化级别的软骨肉瘤组织标本中存在PTHrP/Hedghoge和Wnt通路的异常表达，且与肿瘤细胞的增殖能力有关。重组人PTH细胞因子可以通过作用于并抑制细胞表达primary cilia来促进软骨肉瘤细胞的去分化，并促进Hedghoge和Wnt下游目的基因的激活。过表达Dkk1可以有效抑制PTH所介导的促软骨肉瘤细胞去分化的作用，抑制肿瘤恶性生物学特性。PTHrP/Hedghoge通路和Wnt通路的双向调控可以影响软骨肉瘤细胞去分化进程，而primary cilia可能是潜在的交汇作用位点。.科学意义：PTHrP/Hedghoge及Wnt信号通路均是软骨发育的关键调控通路。本课题研究显示在软骨肉瘤细胞的发育过程中存在二者信号通路的异常表达，同时通过调控二者的交互作用可以影响软骨肉瘤细胞的去分化能力，本课题的研究为进一步研究软骨肉瘤细胞的去分化调控及寻找可能的治疗靶位点提供了一定的理论基础和实验依据。