热休克蛋白47和硒蛋白S低表达协同致内质网应激与大骨节病软骨细胞死亡的作用研究

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 BiP/Grp78，one of Biomarker for Endoplasmic Reticulum (ER) Stress， increased in whole zone of cartilage of Kashin-Beck disease. And ER-resident chaperone proteins Heat-shock protein 47 (HSP47) and selenoprotein S decreased in cartilage of KBD and a rat model of Kashin-Beck disease using T-2 toxin and selenium deficiency conditions.Thus, our hypothesis is that down-regulation of HSP47 and selenoprotein S may be involved in sustained ER stress and initiate chondrocyte death in KBD. Though establishment of decreased expression of HSP47 or/and selenoprotein S in chongdocytes and engeering cartilage models, three specific aims are proposed to address the hypothesis. Specific Aim 1 is identify whether decreased expression of HSP47 involved in ER stress and initiate chondrocyte death and matrix degradation through knock-down/out HSP47 gene expression using real-time PCR and western blot etc. Specific Aim 2 is identify whether decreased expression of selenoprotein S involved in Endoplasmic Reticulum-associated Degradation associated ER stress and chondrocyte death through knock-down/out selenoprotein S gene expression. Specific Aim 3 is identify synergistic effect of decreased expression of HSP47 and selenoprotein S is involved in chondrocyte death through knock-down/out both HSP47 and selenoprotein S gene expression. Specific Aim 4 designed to screen out the abnormal chondrocyte death target moleculars induced by HSP47 and selenoprotein S knockout cells using 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. 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。前期研究发现内质网应激标志物蛋白BiP/Grp78在KBD儿童软骨表达升高,内质网驻留功能蛋白HSP47和硒蛋白S在KBD儿童和低硒条件下T-2毒素中毒大鼠软骨表达减少甚至缺失。提出HSP47和硒蛋白S协同低表达共同激活内质网应激，导致KBD深层软骨基质破坏和细胞死亡的假设。本项目以HSP47和硒蛋白S表达减少为切入点，构建HSP47和硒蛋白S低表达的软骨细胞和组织模型，通过实时定量PCR、Western Blot和基因沉默/敲除等技术，旨在：①阐明HSP47和硒蛋白S低表达致内质网应激与KBD的关系；②明确HSP47或/和硒蛋白S下调表达致软骨细胞死亡和基质降解的作用；③筛选HSP47和硒蛋白S协同低表达致软骨细胞死亡的下游效应靶分子，并进行验证。通过系统研究HSP47和硒蛋白S协同对软骨坏死的作用，为揭示KBD发病机制提供理论依据。

我们前期研究发现，在KBD儿童软骨中，内质网应激标志物（BIP/ GRP78）表达升高，而内质网驻留功能蛋白热休克蛋白47（Heat-shock protein 47, HSP47）和硒蛋白S明显减少甚至缺失，提出HSP47和硒蛋白S异常表达引起的内质网应激在KBD发病中起重要作用的假设。随后，我们在低硒条件下T-2毒素中毒大鼠模型关节软骨以及T-2毒素处理的软骨细胞中也检测到内质网应激相关分子表达水平升高，而内质网应激抑制剂可以部分阻断T-2毒素引起的软骨细胞凋亡和细胞外基质降解，证明内质网应激与KBD发生发展密切相关。在低硒条件下T-2毒素中毒大鼠模型关节软骨及T-2毒素处理的软骨细胞中同样观察到HSP47表达减少，补硒可以部分阻断T-2毒素诱导的HSP47表达减少，因此构建HSP47低表达的肥大软骨细胞，发现HSP47低表达可引起内质网应激，并发生软骨细胞死亡和基质降解。另外，我们通过低硒大鼠模型及细胞模型发现硒蛋白S是一种硒敏感蛋白，低硒营养会使硒蛋白S水平下降。因此构建硒蛋白S低表达的软骨细胞，发现敲低硒蛋白S并不会引起内质网应激，但会加重衣霉素引起的内质网应激。硒蛋白S低表达不影响软骨细胞增殖，也不会引起细胞死亡。硒蛋白S低表达的软骨细胞表现出氧化应激，细胞外基质降解和分化异常。为了进一步验证，我们构建硒蛋白S基因敲除小鼠，并给予T-2毒素处理，发现硒蛋白S基因敲除和T-2毒素协同引起软骨基质降解和分化异常，为低硒条件下T-2毒素中毒的KBD病因学说提供理论依据。最后筛选靶分子发现HSP47低表达可破坏溶酶体酸性环境，通过多种途径致溶酶体功能障碍，可能是导致软骨细胞死亡和基质降解的机制。因此，我们得出结论，KBD的可疑病因低硒条件下T-2毒素中毒通过引起HSP47和硒蛋白S低表达,致软骨细胞内质网应激、溶酶体功能异常、细胞凋亡、氧化应激、基质降解和分化异常。本项目通过系统研究HSP47和硒蛋白S对软骨损伤的影响和信号调控，揭示KBD发病机制，为KBD新药研发提供理论依据。