软骨细胞肥大化是骨骼发育的重要生理过程，既往研究多集中于信号通路及其他因子对软骨细胞肥大化与肢体发育的调控作用，然而其结构基础尚未揭示。研究提示软骨细胞肥大化是大量水分及其他物质蓄积的过程，我们预实验采用活细胞荧光探针染色等发现胞内囊泡融合与聚集极有可能是软骨细胞肥大化的结构基础；单细胞测序等结果提示SNAP23高表达与SNAP25低表达可能是调控这一结构基础的分子机制。因此，本项目围绕SNAP23与SNAP25如何发挥调控作用并影响软骨细胞肥大化及肢体发育这一关键科学问题，体外验证SNAP23与SNAP25对肥大软骨细胞内囊泡融合与聚集的作用；揭示其胞内囊泡融合的分子机制；利用肥大软骨细胞特异性敲除SNAP23和/或敲入SNAP25小鼠，体内研究SNAP23与SNAP25对囊泡融合与聚集、软骨细胞肥大化及肢体发育的影响，以期进一步推动软骨内成骨相关研究的进展和突破。

Chondrocyte hypertrophy is critical for bone development. It has been suggested that chondrocyte hypertrophy was a result of the accumulation of water and other substances. However, previous studies were mainly focused on the effects of regulatory factors, including signaling pathways, on chondrocyte hypertrophy and the limb development, while its structural basis has not been revealed. In our preliminary experiments, we found that the intracellular vesicles fusion and aggregation could be probably the structural basis of chondrocyte hypertrophy. Furthermore, the results of single cell sequencing suggested that the high expression of SNAP23 and low expression of SNAP25 could be the mechanisms regulating the above structural basis. Therefore, this project is aimed to figuring out the key scientific issue of how SNAP23 and SNAP25 regulate the vesicle fusion an aggregation, thereof influencing chondrocyte hypertrophy and limb development. At first, we will validate the effects of SNAP23 and SNAP25 on vesicle fusion and aggregation in hypertrophic chondrocytes by means of labeling with fluorescent probes for live cells et al. in vitro; reveal the molecular mechanisms of vesicle fusion using co-immunoprecipitation; establish hypertrophic chondrocyte specific SNAP23-knockout and/or SNAP25-knockin mice to study the effects of SNAP23 and SNAP25 on vesicle fusion and aggregation, chondrocyte hypertrophy and limb development in vivo. This project will be expected to further promote the progress and breakthroughs in the field of endochondral bone development.