核心蛋白聚糖异常表达对大骨节病软骨坏死的作用与调控机制

The characteristic pathological change of Kashin-Beck disease (KBD) is the occurrance of chondrocyte death in hyaline cartilage including articular cartilage and growth plates.Our previous study showed degradation of proteoglycan in cartilage of KBD, and decreased expression of decorin on whole zone of cartilage of Kashin-Beck disease. Thus, our hypothesis is that decreased expression of decorin induction of abnormal regulation may involved in chondronecrosis in cartilages of KBD. Though establishment of decreased expression of decorin in chongdocytes and engeering cartilage models, three specific aims are proposed to address the hypothesis. Specific Aim 1 is identify whether decreased expression of decorin involved in chondrocyte death and matrix degradation through binding to possible regulatory ligands. Specific Aim 2 designed to screen out the abnormal chondrocyte death target moleculars induced by decorin knockout cells using the The Human Cell Death Pathway Finder RT² Profiler™ PCR Array. And further to identify whether these death target moleculars expression changes with onset and progression in cartilages and serum in children KBD and adult KBD. In Specific Aim 3，we will determine the effects of decreased expression of decorin on target moleculars expression though binding to possible regulatory ligands using Knock-out of decorin chondrocytes. These in vivo and in vitro approaches will provide rationale evidence for elicidating the molecular mechanisms of chondronecrosis in KBD and developing novel targeted therapeutic intervention against KBD progression.

软骨坏死是大骨节病（KBD）的基本病理特征，机制不明。前期研究发现软骨基质降解与KBD发生有关，基质活性小分子核心蛋白聚糖（Decorin）在KBD软骨全层低表达，提出Decorin表达异常介导软骨细胞死亡和胶原纤维降解，在KBD发病中起重要作用的假设。本项目以Decorin异常表达为切入点，构建Decorin低表达的软骨细胞和组织模型；通过：①采用实时定量 PCR、Western Blot和基因沉默/敲除等技术，验证Decorin低表达对细胞存活和基质降解的作用和信号调控机制；②高通量RT-PCR Array技术筛选Decorin低表达致细胞死亡的效应靶分子，并在KBD和动物模型软骨和血液中验证；③采用基因沉默/敲除等技术，明确Decorin低表达对软骨细胞死亡效应分子信号调控机制。通过系统研究Decorin对软骨坏死的影响和信号调控，揭示KBD发病机制，为KBD新药研发提供理论。

我们前期研究发现软骨基质降解与KBD发生有关，基质活性小分子核心蛋白聚糖（Decorin）在KBD软骨全层低表达，提出Decorin表达异常在KBD发病中起重要作用的假设。KBD软骨坏死以侵犯骺生长板和关节软骨的深层软骨细胞为主，KBD的发生涉及软骨细胞死亡、胶原纤维降解。本项目以Decorin异常表达为切入点，发现低硒条件下T-2 毒素组致Decorin蛋白减少，深层未见Decorin表达，和KBD儿童表达情况一致。T-2毒素引起软骨细胞Decorin低表达，构建Decorin低表达的软骨细胞和组织模型，发现Decorin低表达可引起较为明显的软骨破坏，软骨细胞死亡和软骨基质的破坏，因此Decorin低表达与KBD发生发展密切相关。进一步发现Decorin低表达异常调控下游受体分子TGF-β、IGF-IR、integrin α2β1和HGF/C-Met通路，例如Decorin低表达阻断TGF-β/Smad通路，同时激活了HGF/C-Met通路，从而促进基质降解，参与KBD软骨细胞死亡的发生发展。继而敲低软骨细胞 Decorin 基因，应用高通量测序技术筛选差异基因，共筛选出1780差异表达基因，其中下调基因主要富集在细胞黏附、丝裂原活化蛋白激酶信号通路、肿瘤坏死因子通路等，上调基因富集在糖胺聚糖生物合成-硫酸肝素/肝素和丝氨酸和苏氨酸代谢相关通路。最后，我们进行了Decorin低表达的下游软骨细胞死亡相关效应靶分子与KBD的关系，发现软骨细胞下调 Decorin 后，通过下调粘附分子Cadherin 6, Cadherin 11, ICAM-1等表达，抑制细胞增殖和黏附，促进软骨细胞凋亡。明确了坏死性凋亡与KBD的关系，坏死性凋亡抑制剂Nec-1逆转T-2毒素引起的软骨细胞死亡；我们增加了Decorin低表达诱导的软骨细胞分化障碍与KBD的关系研究，发现Decorin参与了软骨细胞的终末分化过程。因此，我们得出结论，Decorin在KBD软骨全层低表达，通过异常调控TGF-β/Smad通路和HGF/C-Met通路促进基质降解，抑制细胞增殖、黏附和分化，促进软骨细胞凋亡和坏死性凋亡，在KBD发病中起重要作用。本项目通过系统研究Decorin对软骨坏死的影响和信号调控，揭示KBD发病机制，为KBD新药研发提供理论。