Name: sequential in vivo imaging of osteogenic stem/progenitor cells during fracture repair
Uploaded: 2014-05-23T13:00:00
Description: Watch this Scientific Journal Video about Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair at JoVE.com

我们感谢C.公园阅读的手稿。这项工作是由NIAMS下奖号码K01AR061434和白血病和淋巴瘤协会奖学金奖（5127-09），以支持DP与健康，以CPL和DTS全国学院授予的内容完全是作者的责任，并且不一定代表美国国立卫生研究院的官方意见。

1，老鼠和预处理注：所有小鼠均保持在无病原体条件和所有的协议获得批准的机构动物护理和使用委员会（IACUC）在马萨诸塞州总医院。所有手术应在无菌条件下用高压灭菌消毒设备的Mx1-Cre重组酶 15进行。，ROSA26-loxP位一站式的loxP-EYFP（罗莎-YFP）和ROSA26-loxP位一站式loxP的tdTomato（罗莎-番茄），分别为从杰克逊实验室购买。 骨钙素-GFP?...

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H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapamycin+affects+early+fracture+healing+in+mice.">Rapamycin affects early fracture healing in mice.</a> Br J Pharmacol. 154, 1055-1062 (2008).</li><li>Maes, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoblast+precursors,+but+not+mature+osteoblasts,+move+into+developing+and+fractured+bones+along+with+invading+blood+vessels.">Osteoblast precursors, but not mature osteoblasts, move into developing and fractured bones along with invading blood vessels.</a> Dev Cell. 19, 329-344 (2010).</li><li>Grcevic, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+fate+mapping+identifies+mesenchymal+progenitor+cells.">In vivo fate mapping identifies mesenchymal progenitor cells.</a> Stem Cells. 30, 187-196 (2012).</li><li>O'Neill, K. 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骨骼干细胞的调控可能是非常重要的定义更好的方法来实现骨骼再生。在细胞水平定量和序列成像一直是技术上的挑战。虽然小鼠长骨骨折模型已被广泛使用，并适合于生物力学研究17，它的深部组织的位置，不均匀断裂大小，软组织损伤，和稳定固定器的应用限制了连续的活体成像。这里，提供了一种通过共聚焦/双光子活体显微镜和鼠标颅骨​​骨折模型的组合来克服这些限制的方法?...

骨退行性疾病和与年龄相关的骨质流失，导致骨质疏松性骨折的高风险已成为公共卫生1的一大挑战。骨的维护是由骨形成的成骨细胞和骨吸收的破骨细胞控制。骨形成细胞的缺陷是与年龄相关的骨丢失和退行性骨疾病2,3的一个主要原因。虽然大量研究都集中在骨折愈合的改善，可靠的药物的发现治愈骨退行性疾病和扭转骨质疏松性骨折的弱点仍然是一个重要的问题。因此，研究骨形成细胞和它们的控制机制在骨再生与修复的来源提供了一种新颖的见解，以加强骨骼的再生和反向骨质流失的疾病。 基于克隆种群的鉴定已经提出了多能间质细胞在骨髓中的存在可能不同黄昏时分为成骨细胞，脂肪细胞和软骨细胞系体外4。近年来，多个研究报告称，骨骼/间充质干细胞（精原干细胞/干细胞）是成骨细胞的天然来源和对骨转换，重构，骨折修复5,6至关重要。此外，我们的谱系追踪研究显示，成熟的成骨细胞有一个意外的半衰期很短（〜60日），并通过他们的干/祖细胞在正常内环境稳定及骨折修复条件6不断补充。然而，干细胞在体内的同一性和细胞是如何起反应，例如破裂损伤，并提供骨形成细胞也不清楚。因此，开发一种方法，能够分析迁移，增殖和内源性精原干细胞/干细胞的分化在生理情况下，它是非常重要的。 骨折修复是通过复杂的阵列调节的多细胞和动态的过程细胞因子和生长因子7。对于骨折研究最流行 ​​的方法是使用具有长骨骨折的动物模型，并通过骨切片和免疫荧光技术8-10来分析骨。此修复过程可以由多个成像技术包括显微CT 11，近红外荧光的12，和化学发光成像13进行监测。然而，每个技术都有一定的局限性，出现了以监测在体内细胞水平的SSC / MSC功能没有有效的方法。最近，共聚焦/双光子活体显微镜已开发和使用，即使在单细胞分辨率来检测移植的肿瘤细胞和造血干细胞在它们的骨髓微环境的上下文中活体动物14。通过这种技术结合了一系列谱系追踪模式，我们能够定义成骨干/祖细胞可以被遗传标记的瞬态交流该粘病毒抵抗 -1（ 的Mx1）启动子和的Mx1诱导祖细胞tivation能保持大多数成熟的成骨细胞的过度时间，但不参与软骨细胞的产生在成年小鼠6。此外，我们证明了的Mx1标记OSPCs供给广大的骨折愈合6个新的成骨细胞。 这里，用骨谱系追踪模式和活体显微镜，一个协议被提供给限定在骨折修复的Mx1 +骨干/祖细胞的体内动力学。该协议提供了连续的成像技术来跟踪搬迁成骨干/祖细胞的成骨折部位和骨原扩张的早期修复过程的定量测量。这种方法可能在多个上下文，包括治疗候选人，以提高骨修复的评价是有用的。

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			<td height="63" style="height:63px;width:216px;">C57BL/6J (H-2b)</td>
			<td style="width:140px;">Jackson Laboratories (Bar Harbor, ME)</td>
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			<td height="42" style="height:42px;width:216px;">Ketamine Hydrochloride Injection</td>
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			<td>Vial: 0.3 mg/ml, Doses: 0.05-0.1 mg/kg</td>
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			<td height="21" style="height:21px;width:216px;">Fine Surgical Scissor</td>
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			<td height="42" style="height:42px;width:216px;">pIpC (Polyinosinic-polycytidylic acid)&#160;&#160;</td>
			<td style="width:140px;">Sigma</td>
			<td style="width:195px;">P0913-50MG</td>
			<td>100 &#956;l (2.5 mg/ml in PBS) for 10 g of mouse</td>
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			<td height="42" style="height:42px;width:216px;">Mai Tai Tunable Ultrafast Lasers</td>
			<td style="width:140px;">Spectra Physics</td>
			<td style="width:195px;"></td>
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			<td height="42" style="height:42px;width:216px;">Dual Calypso 491 + 532 nm DPSS laser</td>
			<td style="width:140px;">Cobolt AB</td>
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			<td height="21" style="height:21px;width:216px;">Radius-635 HeNe laser</td>
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sequential in vivo imaging of osteogenic stem/progenitor cells during fracture repair

骨归连续且高度再生损伤后。成骨细胞的干细胞/祖细胞长期被假设为存在，但在这些细胞的体内示范最近才实现的。这里，所提供的体内成像技术来探讨内源性骨干/祖细胞的作用（OSPCs）及其在骨修复后代。使用骨系细胞追踪模型和颅盖骨诱导微裂缝的活体成像，OSPCs可以直接在受伤后的最初几天，在这在早期修复过程中的关键事件发生时观察到。损伤部位可连续拍摄显露OSPCs搬迁到伤害，增加数量和分化为成骨成骨细胞。这些方法提供了调查的干细胞内在和外在的分子调节剂对骨再生和修复的作用的​​手段。

<html>稳定的长骨骨折模型已经流行在断裂的研究。然而，长骨​​或大骨折模型导致多个组织的损伤，因此，在骨细胞功能的定量测量的限制。我们用针打孔（ 图1A-1C），开发了一种微创损伤（小于1mm直径与最小或没有侵入硬脑膜）上颅骨额骨。我们选择了颅骨额骨用于体内活微裂缝的成像的顶视图，因为这骨有一个平而薄的骨结构与骨髓，允许成像清晰受伤的骨，骨细胞和?...

Watch this Scientific Journal Video about Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair at JoVE.com

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

在骨折愈合的骨祖细胞功能的定量测量需要高分辨率串行成像技术。这里，提供了用于使用活体显微镜和骨谱系追踪以顺序图像和量化修复骨骨折的处理内源性骨干/祖细胞的迁移，增殖和分化的协议。

顺序<em&gt;在体内</em&gt;成骨干/祖细胞在骨折修复的影像学

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in ...

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Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

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