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

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.

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。

椎间盘退行性变(IDD)及其继发的腰腿痛是骨科常见症状，与其相关的误工、致残、以及诊疗康复给社会医疗带来沉重的经济负担。椎间盘脊索快速空泡化和出生后髓核慢性去空泡是椎间盘从产生到退变的整个变化过程，内体形成的Rabs蛋白和介导内体定向转运的微管马达蛋白对脊索产生与胞浆空泡丢失起关键的调控作用。我们实施本课题研究中发现：1.大鼠胚胎脊柱切片观察到椎间盘形成过程中脊索快速空泡化以及椎间盘形成之后髓核缓慢去空泡化现象。2. 脊索快速空泡化产生的膨胀力潜在参与椎间盘形成和形态维持，在脊索被挤压入髓核过程中，脊索鞘逐渐变薄消退，空泡化的脊索极可能与浸润的椎体原基相互对抗而挤压降解脊索鞘。3. 发现自然衰老、纤维环穿刺加速退变组大鼠椎间盘内髓核去空泡化的差异，椎间盘高度逐渐下降，髓核与纤维环分界消失。4. 不同培养时间对脊索样髓核细胞内空泡形态存在差异，髓核细胞培养4天时观察到较多的大而带细胞空泡结构的脊索样髓核细胞，7天时细胞空泡减少并 “纺锤样”改变。5. PCR和WB检测发现培养4天的髓核细胞中Rab32和Lamp1的表达量较7天显著较多，lamp2和Rab5蛋白显著减少，提示内体蛋白参与调控空泡丢失及髓核细胞老化。6.课题实施过程中，我们丰富和拓展研究IDD微环境作用下A20、GATA4、omentin-1、miR-760、细胞外泌体和线粒体分裂融合蛋白等参与调控髓核细胞凋亡、老化和IDD发生发展。综上所述，我们选择与脊索空泡膜形成与消耗密切相关的内体形成与转运信号为研究切入点，从分子水平、细胞水平、动物水平及临床标本水平多层面研究IDD机制，为深化认识椎间盘形成、发育以及退变过程中的关键性细胞学病因和分子病理学机制打开了崭新思路。