Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model.
Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model.
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DOI：
10.3791/55025
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发表时间：
2017
期刊：
影响因子：
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通讯作者：
Tingsheng Yu;C. Winkler
中科院分区：
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作者：
Tingsheng Yu;C. Winkler
成骨细胞与破骨细胞相互作用，协调骨基质的转换和控制骨骼的稳态。青鳉和斑马鱼幼体被广泛用于分析骨形成、退化和修复过程中骨细胞的行为。它们的光学透明度允许荧光标记的骨细胞和结合到矿化骨骼基质的荧光染料的可视化。我们的实验室已经产生了转基因青鳉鱼，表达破骨细胞诱导因子核因子κB配体受体激活剂（RANKL）的热休克诱导启动子的控制下。RANKL的异位表达导致活化的破骨细胞的过量形成，这可以在组织蛋白酶K（ctsk）启动子控制下在具有nlGFP表达的报告细胞系中观察到。RANKL诱导和异位破骨细胞形成导致严重的骨质疏松样表型。在破骨细胞中表达ctsk：nlGFP的复合转基因青鳉品系，以及在早熟成骨细胞中在osterix（osx）启动子控制下表达mCherry的复合转基因青鳉品系，可用于研究两种细胞类型的相互作用。这有助于在骨退化和修复条件下对细胞行为的体内观察。在这里，我们描述了使用该系统来测试一种常用于人类骨质疏松症治疗的药物，并描述了一种实时成像的协议。青鳉模型补充了细胞培养和小鼠的研究，并提供了一个新的系统，在骨骼系统中的药物作用的体内分析。
Bone-forming osteoblasts interact with bone-resorbing osteoclasts to coordinate the turnover of bone matrix and to control skeletal homeostasis. Medaka and zebrafish larvae are widely used to analyze the behavior of bone cells during bone formation, degeneration, and repair. Their optical clarity allows the visualization of fluorescently labeled bone cells and fluorescent dyes bound to the mineralized skeletal matrix. Our lab has generated transgenic medaka fish that express the osteoclast-inducing factor Receptor Activator of Nuclear-factor κB Ligand (RANKL) under the control of a heat shock-inducible promoter. Ectopic expression of RANKL results in the excess formation of activated osteoclasts, which can be visualized in reporter lines with nlGFP expression under the control of the cathepsin K (ctsk) promoter. RANKL induction and ectopic osteoclast formation leads to severe osteoporosis-like phenotypes. Compound transgenic medaka lines that express ctsk:nlGFP in osteoclasts, as well as mCherry under the control of the osterix (osx) promoter in premature osteoblasts, can be used to study the interaction of both cell types. This facilitates the in vivo observation of cellular behavior under conditions of bone degeneration and repair. Here, we describe the use of this system to test a drug commonly used in human osteoporosis therapy and describe a protocol for live imaging. The medaka model complements studies in cell culture and mice, and offers a novel system for the in vivo analysis of drug action in the skeletal system.