IN VIVO IMAGING OF GROWTH PLATE DYNAMICS

描述(申请人提供)：儿童骨骼生长是由长骨末端软骨生长板中的软骨内骨化引起的。在过去的十年中，对转基因结构的分析已经成为研究内分泌、旁分泌和自分泌调节因子在软骨细胞分化级联过程中作用的最有效的方法。然而，尽管在描述定义骨骼生长及其调控回路的细胞和分子事件方面取得了重大进展，但对于内分泌(和潜在的旁分泌)调节因子到达生长板软骨细胞所需的血管通路的生理学，以及这些分子如何在空间上不同的生长板基质中移动，人们知之甚少。这在很大程度上反映了缺乏在活体动物的实时条件下研究这些问题的方法。这对研究生长板尤其具有挑战性，生长板有三个概念上不同的血液供应：骨骺、干骺端和软骨膜(包括一个环状血管和一个神经丛)。 我们将利用多光子显微镜(MPM)对小鼠胫骨近端生长板进行多小时活体成像，从而应用一种新的方法来解决这些问题。我们的实验系统是麻醉的4-5周大的转基因小鼠，带有连接到II型胶原启动子的绿色荧光蛋白(Col II/GFP)。具体目标概括如下： 通过直接成像静脉注射的荧光示踪剂的到达(从右旋糖苷开始，但转移到已知的具有生物学意义的软骨细胞分化调节因子)，来测试不同大小的分子进入生长板的速率不同的假设。 为了验证不同分子量的示踪剂通过不同的血管途径到达生长板，并在不同的基质亚室中在生长板内移动的假设。对于骨痂血管系统，分析将包括次级骨化中心完全发育之前的时间点。 实时成像干骺端软骨骨交界处的细胞周转事件，作为与骨延长率数据相关联的“内部时钟”。 这些分析与对生长异常的理解有关，例如过早闭合的创伤和导致过度生长的骨膜破裂。对于生长板的血管路径和分子在生长板内的运动的详细功能知识是必不可少的，因为人们认为将治疗药物靶向生长板是必要的。

DESCRIPTION (provided by applicant): Bone growth in children occurs by endochondral ossification in cartilaginous growth plates at the ends of long bones. In the last decade, analysis of transgenic constructs has become the single most powerful approach for the study of endocrine, paracrine and autocrine regulators acting during the chondrocytic differentiation cascade. However, despite major advances in delineating the cellular and molecular events that define skeletal growth and its regulatory circuitry, relatively little is known about the physiology of the vascular access that endocrine (and potentially paracrine) regulators require to reach growth plate chondrocytes, and how these molecules move within the spatially heterogeneous growth plate matrix. To a large extent this reflects a lack of methods to study these questions under real time conditions in living animals. This is particularly challenging for studying the growth plate, which has three conceptually different blood supplies: epiphyseal, metaphyseal, and perichondrial (including a ring vessel and a plexus.) We will apply a novel approach to these questions by utilizing multiphoton microscopy (MPM) for multi-hour in vivo imaging of the murine proximal tibial growth plate. Our experimental system is anesthetized 4-5-week-old transgenic mice with green fluorescent protein linked to the collagen II promoter (Col II/GFP). The specific aims are summarized as: To test the hypothesis that the rate of delivery into the growth plate varies for molecules of different size, by directly imaging the arrival of IV injected fluorescent tracers (starting with dextrans but moving to known biologically significant regulators of chondrocytic differentiation.) To test the hypothesis that tracers of different molecular weight reach the growth plate through different vascular routes and travel within the growth plate in different matrix subcompartments. For the epiphyseal vasculature the analysis will include time points prior to full development of the secondary center of ossification. To image cellular turnover events at the metaphyseal chondroosseous junction in real time, as an "internal clock" for correlation with data on rates of bone elongation. These analyses are relevant to the understanding of growth aberrations such as trauma with premature closure and disruption of the periosteum leading to overgrowth. Detailed functional knowledge of vascular routes to the growth plate, and movement of molecules within the growth plate, is essential as thought is given to targeting therapeutic agents to the growth plate.