SNAP23/25调节胞内囊泡融合与聚集促进软骨细胞肥大化的作用及其机制研究

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.

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。

软骨细胞肥大化在软骨内成骨发育、骨性关节炎等运动系统生理病理过程中是最关键的细胞形态学及生物学演变之一。本项目前期研究发现胞内囊泡融合与聚集是软骨细胞肥大化的结构基础。为研究其分子机制，本项目首先采用小鼠原代生长板细胞建立并验证体外软骨细胞肥大化模型；同时检测了SNAREs家族关键分子的表达规律与体内一致；继而，我们构建了SNAP23、SNAP25过表达质粒，利用上述体外细胞模型，检测SNAP23、SNAP25对囊泡形成、融合等的影响；同时探索了囊泡的来源及规律，证实肥大软骨细胞内囊泡是内质网逐步囊泡化并融合而成；最后，采用单细胞测序及免疫荧光染色等实验，发现与关节软骨细胞相比，肥大软骨细胞钠钾等离子通道特异性高表达，提示其肥大化过程中离子物质的细胞内运输活跃；此外，介导内质网融合的关键分子Atl1与内质网囊泡化及聚集活跃度呈正相关，可能是另一关键调节机制；上述分子与SNAREs协同调控肥大软骨细胞内质网囊泡化与融合的作用及机制，是我们进一步的研究内容。