Formation of the intervertebral disk: a molecular approach

据估计，三分之二的美国人在一生中至少会经历一次背痛。背痛影响超过一半的65岁以上的人，在美国每年治疗背痛花费500 - 1000亿美元。大多数背痛被认为是由于椎间盘损伤造成的，椎间盘位于每个椎骨之间。目前还没有治疗因物理损伤而受损或已退化的椎间盘的方法。母公司R 01提案的目标是确定椎间盘形成的细胞和分子机制，长期目标是开发基于细胞或蛋白质的椎间盘退变疗法。Harfe实验室在发育生物学和遗传学方面拥有丰富的专业知识，这使我们能够快速识别胚胎脊索作为形成成熟椎间盘的整个中间部分的细胞群，称为髓核（这是父母资助的目标之一，这项工作现已发表）。这是椎间盘的这个区域，通常在患有严重背痛的患者中受损。在这个建议中，我们描述了试点实验，以开发一种基于细胞的治疗方法，使用纯化的脊索细胞和椎间盘退变模型来治愈受损或退化的椎间盘。该项目是佛罗里达大学的Harfe实验室和宾夕法尼亚大学的Elliott实验室之间的一项新合作。Elliott实验室在椎间盘机械功能和退变、椎间盘退变动物模型和椎间盘组织工程方面拥有丰富的专业知识。这两个研究小组共同拥有独特的能力，可以将我们对髓核发育的理解转化为椎间盘退变的细胞疗法。使用通常形成髓核的细胞来治愈受损椎间盘的治疗机会被确定为母基金5年范围之外的未来目标。由于我们最近对髓核前体细胞的鉴定，现在可以开始实验来直接测试这些细胞的可能治疗作用。与Elliott博士的跨学科合作关系通过立即将Harfe博士发现的对椎间盘形成的发育机制的基本理解转化为治疗椎间盘退变，显著提高了母基金的范围。在这个提议中，我们将开始通过首先将纯化的脊索细胞注射到正常小鼠的成年髓核中来测试脊索细胞的治疗潜力（目的1）。我们最近令人兴奋的发现，即所有存在于成熟小鼠髓核中的细胞都来自胚胎脊索，这表明脊索细胞可能能够发挥椎间盘干细胞的作用。为了测试纯化的脊索细胞是否可以分化为成熟髓核中发现的所有细胞类型，我们建议将这些细胞（用LacZ标记）注射到成年小鼠髓核中。它们存活、增殖和形成所有髓核细胞类型的能力将使用分子标记来确定。

DESCRIPTION (provided by applicant): It has been estimated that two-thirds of Americans will experience at least one episode of back pain in their lifetime. Back pain affects more than half of people over the age of 65 and the treatment of back pain costs 50- 100 billion dollars per year in the United States. Most back pain is thought to result from damage to the intervertebral disc, which are located between each vertebra. Presently there is no cure for intervertebral discs that have been damaged either by physical injury or have degenerated. The goal of the parent R01 proposal is to determine the cellular and molecular mechanism(s) responsible for the formation of the intervertebral discs with a long-term goal to develop cell or protein-based therapies for disc degeneration. The Harfe laboratory has extensive expertise in developmental biology and genetics, which allowed us to quickly identify the embryonic notochord as the cell population that forms the entire middle part of the mature disc called the nucleus pulposus (this was one of the aims of the parent grant and this work has now been published). It is this region of the disc that is usually damaged in patients that have severe back pain. In this proposal, we describe pilot experiments to develop a cell-based therapy to heal damaged or degrading discs using purified notochord cells and a model of disc degeneration. This project is a new collaboration between the Harfe laboratory at the University of Florida and the Elliott laboratory at the University of Pennsylvania. The Elliott laboratory has extensive expertise in disc mechanical function and degeneration, animal models of disc degeneration, and disc tissue engineering. Together, the two groups are uniquely equipped to translate our understanding of nucleus pulposus development into cell-based therapies for disc degeneration. The therapeutic opportunity of using cells that normally form the nucleus pulposus to heal damaged discs was identified as a future goal, beyond the 5-year scope, of the parent grant. Due to our recent identification of nucleus pulposus precursor cells it is now possible to begin experiments to directly test possible therapeutic roles for these cells. The interdisciplinary partnership with Dr. Elliott significantly enhances the scope of the parent grant by immediately translating the basic understanding of the developmental mechanism responsible for disc formation discovered by Dr. Harfe to treating disc degeneration. In this proposal we will begin to test the therapeutic potential of notochord cells by first injecting purified notochord cells into the adult nucleus pulposus of normal mice (Aim 1). Our recent exciting discovery that all cells present in the mature mouse nucleus pulposus are derived from the embryonic notochord suggests that notochord cells may be able to function as disc stem cells. To test if purified notochord cells can differentiate into all cell types found in the mature nucleus pulposus we propose to inject these cells, marked with LacZ, into the adult mouse nucleus pulposus. Their ability to survive, proliferates, and form all nucleus pulposus cell types will be determined using molecular markers.