DDIT3对软骨内成骨的调控作用及相关机制研究

Bone tissue engineering based on endochondral ossification provides a new way to repair large bone defect. However, the success of this approach is hindered by core degradation and an uneven distribution of bone mineral throughout the construct. DNA damage-inducible transcript 3 (DDIT3) is a member of the C/EBP family of transcription factors that participates in immune reactions and cell differentiation. As an apoptotic transcription factor, DDIT3 expression can be induced in response to endoplasmic reticulum stress, and plays a crucial role in pathogenesis of inflammation and cell death during chronic stress. Considering the importance of apoptosis in chondrocytes differentiation, we hypothesize that, by studying the function and mechanism of DDIT3, it can control the differentiation of chondrocyte as well as the following endochondral ossification. To evaluate the effect of DDIT3 on endochondral ossification in various stages, ATDC5 cells with enhanced or reduced DDIT3 function driven by different promoters are developed in vitro, and then the conditional knockout mice are employed to investigate the function of DDIT3 in endochondral ossification in vivo. Finally, through gene regulation, the chondrogenic differentiation and the following endochondral ossification are observed. We expect to illuminate the mechanism of DDIT3’s effect on the chondrocytes differentiation and endochondral ossification. This study will provide us theoretical basis to regulate the bone tissue repair process reasonably, to promote angiogenesis more effectively, and finally to boost new bone formation in the large bone defect.

DDIT3是内质网应激通路下游重要的转录因子，能够介导细胞死亡，鉴于肥大软骨细胞凋亡在软骨内成骨中的重要作用，结合前期研究基础，我们推测：DDIT3很可能是调控软骨细胞分化的重要转录因子，通过直接或间接地抑制SIRT1的表达和活性，对软骨发育及软骨内成骨起负调控作用。抑制SIRT1一方面下调了Dvl蛋白，从而抑制了Wnt通路；另一方面SIRT1的下降减少了FoxO1的乙酰化，抑制了自噬通路。此外，抑制SIRT1还可能抑制SOD2蛋白，放大ROS作用，促进软骨细胞炎症和凋亡。为验证这一假设，我们首先检验DDIT3与软骨内成骨各个阶段的相关性，观察DDIT3对转基因小鼠软骨内成骨的影响；然后探讨DDIT3调控软骨向分化的机制；最后联合支架材料，了解DDIT3影响软骨内成骨的效果。我们期望通过阐明DDIT3对软骨细胞分化及软骨内成骨的影响和作用机制，可能调控经由软骨内成骨的大面积骨缺损修复过程。

本项目从整体、细胞和分子水平阐明了DDIT3在内质网应激引起的软骨异常、颞下颌关节炎、炎性颅骨骨溶解、牙周炎及牙髓炎等疾病发生发展中的调控作用及具体机制。研究结果显示：DDIT3通过SIRT1-AKT通路促进软骨细胞自噬，提示DDIT3可能在软骨发育及软骨内成骨中起重要作用；DDIT3在内质网应激状态下通过AMPKα-SIRT1通路抑制软骨细胞分化，并且在DDIT3敲除小鼠体内可减缓内质网应激介导的软骨厚度降低和成软骨抑制；DDIT3在炎症状态下通过巨噬细胞焦亡的旁分泌作用抑制软骨细胞的细胞外基质合成代谢，从而可能加速颞下颌关节炎的进展；DDIT3负向调控破骨细胞的形成、分化和功能；DDIT3敲除通过促进破骨细胞的生成和功能加重牙周炎中的骨吸收；DDIT3通过抑制线粒体自噬促进原代成骨细胞焦亡；DDIT3通过NF-κB通路调控IDH1和成牙骨质细胞矿化；DDIT3通过促进巨噬细胞M1型极化并抑制M2型极化加重牙髓炎炎症发展。以上结果充分表明DDIT3在软骨及机体硬组织发育、骨代谢和炎症损伤中发挥重要作用，涉及多种细胞内的多个重要信号通路。该项目为DDIT3在软骨、成骨、破骨和炎症相关疾病中的分子机制和基因治疗提供了理论依据。