Molecular imaging of the skeleton: Quantitative real-time bioluminescence monitoring gene expression in bone repair and development
Molecular imaging of the skeleton: Quantitative real-time bioluminescence monitoring gene expression in bone repair and development
复制标题
DOI：
10.1359/jbmr.2003.18.3.570
复制
发表时间：
2003-03-01
影响因子：
6.2
通讯作者：
Gazit, D
中科院分区：
文献类型：
--
作者：
Bar, I;Zilberman, Y;Gazit, D
无创地监测体内基因表达是有效基因治疗系统的关键问题。迄今为止，还没有足够的分子成像技术来定量监测骨骼发育和修复中的体内基因表达。本研究的目的是利用实时分子成像系统监测骨骼发育和修复中的基因表达，该系统可定量且无创地检测体内生物发光。我们的实验模型由在人骨钙素（hOC）启动子调控下携带荧光素酶标记基因的转基因小鼠组成。应用新型光检测冷却电荷耦合器件（CCCD）相机来监测荧光素酶表达。在体外，从转基因小鼠骨髓中分离的间充质干细胞（MSC）表现出 hOC 启动子调节，通过与其成骨分化相关的荧光素酶表达检测到。在1周至1.5年的发育过程中，转基因小鼠在广泛的骨骼器官中表现出转基因表达，包括颅骨、椎骨、尾巴和四肢，大部分骨骼器官在第1周达到峰值。在两种骨骼修复模型（骨折和骨髓消融）中，无创 CCCD 系统显示术后 6 天荧光素酶表达达到峰值。所有定量、无创、实时 CCCD 测量均与荧光素酶生化测定和荧光素酶免疫组织化学相关，证明肥大软骨细胞和小梁成骨细胞中存在荧光素酶表达。我们的研究首次表明：（1）CCCD检测系统是一种可靠的体内骨骼定量基因检测工具，（2）大多数骨骼部位受hOC启动子调控的荧光素酶表达随着年龄的增长而显着下降，（3）实时、定量、无创地揭示小鼠骨骼发育和修复过程中hOC调控的动态。
Monitoring gene expression in vivo, noninvasively, is a critical issue in effective gene therapy systems. To date, there are no adequate molecular imaging techniques, which quantitatively monitor gene expression in vivo in skeletal development and repair. The aim of this study was to monitor gene expression in skeletal development and repair, using a real-time molecular imaging system, which quantitatively and noninvasively detects bioluminescence in vivo. Our experimental model consisted of transgenic mice harboring the luciferase marker gene under the regulation of the human osteocalcin (hOC) promoter. A new light detection cooled charge coupled device (CCCD) camera was applied to monitor luciferase expression. In vitro, mesenchymal stem cells (MSCs) isolated from bone marrow of transgenic mice exhibited hOC promoter regulation, detected by luciferase expression that correlated with their osteogenic differentiation. During development from 1 week to 1.5 years, transgenic mice exhibited transgene expression in a wide spectrum of skeletal organs, including calvaria, vertebra, tail, and limbs, reaching a peak at I week in most of the skeletal organs. In two skeletal repair models, bone fracture and marrow ablation, the noninvasive CCCD system revealed a peak of luciferase expression at 6 days postsurgery. All quantitative, noninvasive, real-time CCCD measurements correlated with a luciferase biochemical assay and luciferase immunohistochemistry, which demonstrated luciferase expression in hypertrophic chondrocytes and trabecular osteoblasts. Our studies show for the first time (1) the CCCD detection system is a reliable quantitative gene detection tool for the skeleton in vivo, (2) expression of luciferase regulated by the hOC promoter is significantly decreased with age in most skeletal sites, and (3) the dynamics of hOC regulation during mice skeletal development and repair in real time, quantitatively and noninvasively.