内体形成与转运信号调控在脊索快速空泡化与髓核慢性去空泡化中的效应机制研究

胞浆大量空泡是脊索样髓核细胞最具特征性的形态学标志。内体形成与转运信号调控是细胞产生和利用浆膜结构的核心机制。本课题以椎间盘形成过程中脊索快速空泡化和出生后髓核慢性去空泡现象为研究切入点，首先通过一系列模型动物诱导和细胞分子生物学实验，探讨参与内体形成的Rabs蛋白和介导内体定向转运的微管马达蛋白在脊索产生与消耗胞浆空泡背后的调控规律，筛选出关键信号分子；继而通过观察低氧、偏酸、高渗、营养匮乏等椎间盘微环境对原代脊索样髓核细胞的影响，细化认识脊索样髓核细胞空泡丢失的潜在病因与信号机理；最终通过脊索条件性敲除rab32基因构建脊索空泡化受限的模式小鼠，从力学支持角度拓展认识胚胎期脊索快速空泡化对椎间盘正常形成与形态维持的潜在效应。本研究将从内体形成与转运信号调控角度深化认识椎间盘形成、发育以及退变过程中的细胞学基础和分子病理学机制，同时为进一步拓展基于脊索样髓核细胞的生物学修复打开崭新思路。

The morphology and functionality of notochord cells are spatiotemporally regulated during intervertebral disc (IVD) formation, development, ageing and degeneration.To date, the molecular mechanism underlying these dynamic cellular changes remains elusive. Compared with other musculoskeletal cells, the notochordal nucleus pulposus cells (NNPCs) are unique in exhibiting large and abundant cytoplasmic vacuoles, which are membrane-bound and generated through a fast vacuolation process early in embryogenesis. Postnatally, these cytoplasmic vacuoles will undergo a gradual de-vacuolation and be barely detected within aged and degenerated nuclues pulposus (NP). While cytoplasmic vesicles have been known to be supplied mainly by post-Golgi biosynthetic trafficking and endocytic trafficking of the internalized membrane, more studies are needed to expand our understanding of the dynamic regulation and signaling basis of notochord vacuolation and de-vacuolation..In this present study, the expression of Rabs proteins of small GTPases family and the kinesins and dynein components of microtubules will be selectively evaluated in the forming vertebral column of rat embyos, aiming to identify the key endocytic regulators of notochord vacuolation. To decode the endocytic pathways regulating de-vacuolation, the gradual loss of notochord vacuoles will be studied in the IVD of natural ageing rats, whereas the accelerted de-vacuolation of NP will be investigated within degenerated IVD induced by annulus stabbing and unphysiological mechanical loading. These innovative studies are expected to provide new clues to better understand the generation and exhaustion of notochord vacuoles, from the perspective of endocytic pathway and trafficking..Unlike other musculoskeletal tissues, the IVD is also unique in its microenvironment, characterized by hypoxia, increased acidity, hypertonicity, nutritional deficiency, as well as cyclic mechanical loading. Although evidence is mounting that the morphology and viability of disc cells are closely regulated by disc microenvironment, it remains unclear how these biochemical and mechanical factors work on the endocytic pathways and regulate the notochord vacuoles. In this study, the vacuoled NNPCs will be isolated and cultured under conditions simulating the characteristic disc microenvironment. By comparing the de-vacuolation process and endocytic regulation in the cultured NNPCs under different conditions, our understanding of the pathogenesis and signaling cascade that underlies the loss of notochord vacuoles will be enhanced.Based on our previous findings that the generation of IVD is accompanied by thinning and vanishing of notochord sheath, possibility can not be ruled out that the notochord-derived tension counteracts the pushing force of growing vertebrae.For validation, we will conditionally deleted rab32 in notochordal cells and investigate how loss of this late endosomal trafficking gene impacts notochord vacuolation, IVD formation, and postnatal maintenance of the geometry shape of normal spine. These studies will help to identify a mechanically supportive role of the vacuolating notochord that probably contributes to the construction of a disc-connected spine..Conclusively, our study will add new evidence to support that the notochord vacuoles are active and versatile organelles for both embryonic notochord cells and postnatal NNPCs. By unfolding the signaling pathways that mediate the formation and function of notochord vacuoles, the age-related exhaustion of NNPCs and particularly the premature de-vacuolation and degeneration in human IVD could be better understood. Targeting de-vacuolation or preserving vacuolating signals is expected to provide novel clues to decelerate IVD degeneration.