Name: tracking dynamics of muscle engraftment in small animals by in vivo fluorescent imaging
Uploaded: 2009-09-21T15:00:00
Duration: 8 min 15 s
Description: Watch this Scientific Journal Video about Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging at JoVE.com

杨的Z和Y王的工作才得以从NIAMS /美国国立卫生研究院授予K02 ​​AR051181，肌肉萎缩症协会和哈佛干细胞研究所的王为Y的赠款。曾的Q和X徐的工作是从布里格姆与妇女医院的光学成像实验室授予的程序授予的支持。作者想感谢来自麻醉科，三围，刘雯在细胞工作的技术帮助。

细胞制备<ol><li>卫星细胞的分离<ol style="list-style-type:lower-alpha;"><li>在麻醉下，绿色荧光蛋白转基因小鼠（C57BL / KA -βactin- EGFP）的四肢肌肉都被删除。切割切成小块后，卫星细胞的酶分离剁碎组织孵育1小时与2％dispase胶原酶溶液在37 ° C </li><li>中含火腿的F - 10和20％胎牛血清的增长媒体的酶消化，游离肌肉，反复用巴斯德吸液管的研制过程。这些细胞是通过细胞过滤器（Ø70 - 100微米），过滤，然后镀上了1小时的非涂布菜在37 ° C </li><li>轻轻地取出上清液胶原蛋白涂层菜和板材他们。重复此步骤，1小时后，以净化成肌细胞。 （板前） </li><li>媒体和板的细胞及时更换。最终纯化的成肌细胞是培养在培养基中含有火腿架​​F - 10，20％胎牛血清，100 U / ml青霉素和100μg/ mL链霉素（生长介质），在37℃，5％CO2，95％的空气。 </li></ol> </li><li>文化和扩大与另外10毫微克/毫升的基本FGF生长介质中的主要成肌细胞。 </li><li>对于所有在体内实验中，我们注入5X10 每 5电讯管理局局长肌肉细胞。 </li><li>收获的成肌细胞时，我们用0.25％胰蛋白酶/ EDTA分离​​的细胞，用PBS洗两次。手机号码与一个血球计数和细胞中的HBSS悬浮5X10 4个细胞/μL浓度。在HBSS中浓缩的细胞被放置在冰和采集后1小时内注射。 </li></ol> 在活体成像<ol><li>男MDX在4-6周的年龄小鼠均购自杰克逊实验室，并保持在畜舍设施。 </li><li>经过约一个星期的住宿环境，准备与培养的细胞植入小鼠。 </li><li>细胞移植前，鼠标是由腹腔注射氯胺酮（100毫克/公斤）和甲苯噻嗪（10毫克/公斤）的混合物（IP）麻醉。然后周围的TA肌肉面积的头发被删除申请奈尔的腿，并等待30秒，擦拭干净，用蒸馏水。 </li><li>当鼠标仍然是在麻醉状态下，5 × 10 5细胞中加入10μl的HBSS慢慢注入到每个助教肌肉在3分，使用30号针头的。 </li><li>第4步后，鼠标可以定期在活体荧光成像成像。荧光成像站NightOwl LB981（贝特霍尔德科技公司），配备具有较高的敏感电荷耦合相机，是用来获取图像从小鼠的阻碍腿。 </li><li> 在活体成像的日子，我们先检查对TA肌肉毛发再生。如果有必要，我们再次申请奈尔删除如上所述的头发后，麻醉与氯胺酮和甲苯噻嗪的混合鼠标。 </li><li>尽管鼠标是在麻醉状态下，我们把鼠标俯卧在一块黑色非荧光纸（斯特拉斯莫尔黑Artagain纸）。我们要充分暴露电讯局长的肌肉，为更好地成像，胶带纸的后爪。 </li><li>我们放置在鼠标的成像室和成像相机领域的中心位置后腿。我们先转出的废气排放过滤器采取了传统的灰度腿与一个5秒的曝光时间的摄影图片。调整鼠标和相机如果有必要的重点的位置。成像参数，包括曝光时间（1250毫秒），试样高度（0.9厘米）等在实验过程中的不断。 </li><li>然后我们在适当的排放过滤器切换为绿色荧光蛋白的波长。我们采取与荧光图像的曝光时间1250毫秒和1分级。成像后，鼠标是从成像室，一个笼子里等待恢复。 </li><li>进行分析，我们绘制的荧光信号的感兴趣区域（ROI）在光子每秒来衡量它们的强度。我们还可以测量作为另一个量化值的像素数量方面的投资回报率的大小。成像实验可以重复每周或更频繁地过了几个月，如果需要的话，获得的纵向数据。 </li></ol>在实验结束时，鼠标是安乐死和TA肌肉组织学分析的收获。

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我们建立了一个可靠和稳定的成像的平台，在跟踪主机骨骼肌在这项研究中的荧光标记的细胞移植。 GFP标记GFP转基因小鼠成肌细胞代表一个类型的成人干细胞，将细胞疗法在未来的候选人。为获得高质量的图像，尤其是进行定量分析，包括细胞数量，细胞注射，背景清晰，成像的位置和机床参数是很重要的一个恒定的操纵。 荧光成像，因为它是在生物医学研究中的许多应用非侵入性的，易于使用，具有高吞吐量。此外，荧光成像技术，可以提供一个基于光子计数，但由于散射，衰减和吸收的定量测量，光无法穿透组织在大的距离，这是该技术的缺点。努力正在使用红色或近红外记者，以增加光线的穿透深度。 荧光成像的另一个优点是，它是翻译的诊所，如果特定的荧光记者批准供人类使用。荧光成像与MRI，CT和PET相比，具有很高的灵活性，因为它不需要昂贵的硬件和显像剂。其中一个例子是基于荧光内窥镜和微型内窥镜已在诊所使用。通过与其他方式相结合的荧光成像技术，我们期望达到的能力，获取解剖和功能有关的基本生物过程的信息。

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tracking dynamics of muscle engraftment in small animals by in vivo fluorescent imaging

肌营养不良症是一组肌肉收缩肌肉细胞逐渐丧失为特征的退行性疾病。目前，有没有可用的治疗服务。在干细胞生物学的最新进展，有有效的细胞为基础的疗法发展产生了新的希望治疗这些疾病。各类成肌肉营养不良的动物模型的荧光蛋白标记的干细胞移植已被广泛用于在现场。一个有能力的非侵入性技术来追踪移植细胞的命运，纵向可以进一步推动我们的能力，更准确，更有效地评估肌肉细胞移植植入。 在本研究中，我们表明， 在体内荧光成像技术是一种用于跟踪移植GFP（绿色荧光蛋白）标记的细胞在小鼠骨骼肌的敏感和可靠的方法。尽管由于使用外部光激发荧光蛋白所必需的有关背景的关​​注，我们发现，使用两种裸鼠或消除脱毛试剂头发这个问题被淘汰。使用CCD摄像头，可以检测到的荧光信号，在胫前局长（电讯局长）肌肉，注射后5 × 10 5细胞无论是绿色荧光蛋白转基因小鼠的eGFP转成肌细胞文化。如较表浅的肌肉，趾长伸肌（EDL），注射较少的细胞产生的检测信号。可以测量信号强度和利息率（ROI）的地区为每秒发出的光子数量量化。由于采集到的图像显示清晰的界限划定栽种面积，投资回报率的大小以及可以测量。如果腿每次定位始终，每秒每肌肉的投资回报率的大小的光子总数的变化反映在嫁接细胞数量的变化和栽种面积的大小。因此，随着时间的推移，在相同的肌肉变化是可以量化的 。体内荧光成像技术已经被主要用于跟踪tumorogenic细胞的生长，我们的研究表明，它是一个强大的工具，使我们能够追踪移植的干细胞的命运。

Watch this Scientific Journal Video about Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging at JoVE.com

Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging

我们描述了一个<em&gt;在体内</em&gt;荧光成像协议监测GFP标记成肌细胞后，移植到健康和营养不良小鼠的骨骼肌肌肉再生。该协议可以适应其他肌肉条件，以及研究其他类型的细胞移植和肌肉再生。

小动物的肌肉植入动态跟踪在体内荧光成像

Muscular dystrophies are a group of degenerative muscle diseases characterized by progressive loss of contractile muscle cells. Currently, there is no ...

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Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging

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