没有资金申报。

所描述的骨骼肌过程遵循阿巴拉契亚州立大学伦理审查委员会的指导方针。 注:该小组由操作者（博士或MD训练的活检技术），和至少一个，但理想的是，三个或更多个技术员。操作员负责进行及监督骨骼肌活检过程的各个方面。一技术员（技术员＃1）是立即用于辅助操作者肌肉提取的所有方面负责。这包括在"非无菌手"和施加抽吸，用无菌注射器。第二技师（技师＃2）在主题和按摩主体的对面站的过程中，尽量减少受试者的焦虑谈话从事。第三技师（技师＃3）是负责处理和加工的骨骼肌样品。下游的分析方法决定多少乳房清洁度有理技术人员所需要的加工/制备骨骼肌样品。 （一）除准备<ol><li>收集，准备和组织的肌肉活检程序（ 表1）中指定的材料。去污，消毒BERGSTROM针（外套管和内套管），并根据标准实践16的柱塞。 </li><li>回顾与主体的知情同意书。在同意参加在阿巴拉契亚州立大学使用形式的骨骼肌活检部分在网上补一。 <ol><li>聊聊肌肉活检过程中，重点放在程序的可能存在的风险，备皮，应用麻醉剂，通常感觉的一般方面的问题。指示须仔细阅读同意参加表格并签字确认，这项研究的设计和程序的理解。 </li></ol></li><li>确认标ubject是不过敏的"___坚"型药物（ 如利多卡因）。 </li><li>指示受仰卧在软垫表暴露大腿。将两个一次性吸收护垫与塑料底板主体的腿下。用脚后跟下一个小毛巾卷，使得膝盖完全伸展（升高约1cm），放置在一个轻松的方式的腿部，从而把股外侧肌在缩短的位置。 </li><li>有操作指示受试者对瞬间收缩的露出大腿肌肉，使得活检部位可以显现。注意:股外侧肌活检部位只是前阔筋膜（illiotibial带），大约三分之一的髌骨的顶部和大转子7之间的距离。 </li><li>刚才下面的操作大关（约0.5厘米）的切口部位用细点永久性标记。 </li><li>在操作员确定大致皮褶厚度由PINC兴的皮褶。 </li><li>有运营商和技术人员洗手用肥皂和温水和唐一次性手套。 </li><li>有运营商从区域去除毛〜15公分×15厘米左右的活检部位（经快船）。注意:从活检部位剪切毛发防止头发从进入切口的过程中和结束时，和脱毛允许磁带关闭和皮肤（见3.4）之间的更好的接触。 </li><li>有运营商棉签消毒面积预浸泡局部消毒剂（如碘伏或洗必泰科目碘过敏/贝类）。开始在中心和同心圆朝向剪切区域的外边缘的工作。至少，有一个新的预先浸泡棉签每次重复两次以上。 </li></ol> 2，活检程序<ol><li>有运营商去除一次性手套，用肥皂和温水洗手，并穿上无菌手术GLoves使用无菌技术。 </li><li>有技术＃1存在于无菌窗孔被单给操作者。有运营商，采用无菌技术，将覆盖在活检部位以保持无菌区。 </li><li>有技术人员＃1目前运营商有5毫升注射器配有21G针头，同时保持无菌操作。清洁利多卡因小瓶用酒精棉签的顶端。 <ol><li>有运营商立即撤除5毫升利多卡因。从注射器中取出21克针，并丢弃针头插入锐器容器中。 </li><li>有技术人员＃1目前有一个1英寸半地下25针的操作，同时保持无菌操作。有操作的地方的1半英寸的针入注射器并从注射器排空气泡。 </li></ol></li><li>有技术人员＃1喷氯乙烷在切口部位（约0.5厘米以上，对皮肤的不褪色墨水标记），直到皮肤出现"灼"。 <ol><li>哈已经操作员插入的针大致水平的皮肤进入真皮，抽吸针，然后渗透用〜100μl的利多卡因，以产生一个"气泡"2-4毫米的直径。 <ol><li>有操作抽吸针（略撤回注射器的柱塞），以确认针头尚未放置在血管中。如果血液中出现的注射器，抽出针，丢弃在一个锐器容器，并在2.3重新开始。 </li><li>有操作者预先将针插入皮下组织，抽吸针，然后渗透用〜1毫升利多卡因的组织以形成气泡。一旦水泡已经消退，将在利多卡因装注射器的1½寸毫针垂直插入切口部位，停止向浅筋膜。注:如有可能最初注射利多卡因（类似于蜜蜂蜇）的时感到轻微短暂的刺痛感。 </li></ol></li><li>确保OPERAT或者没有渗透到肌肉与lidocoine因为它是myotoxic 17-19。 </li><li>有运营商抽吸针，然后慢慢注入利多卡因的剩余4毫升同时取出从大腿针。将无菌纱布注射部位，并让受放松，而局部麻醉生效。 </li></ol></li><li>经过2-3分钟，具有技师＃1本操作手术刀，同时保持无菌操作。 <ol><li>有操作者轻轻探查活检部位用手术刀的前端，以确认该区域的麻醉。如果有必要，又注入3-4毫升利多卡因进入切口部位，见2.4.1.2。 </li></ol></li><li>有操作员通过皮肤和皮下组织（〜2-3毫米以上的墨水标记）平行于股骨作出的直1厘米的切口。 <ol><li>有运营商将手术刀通过更深层次的筋膜进入肌肉做一个切口。在每个方向上，一旦做到这一点<html> 。注:如有可能会感到刺痛或压力的更深层次的剪裁，如果手术刀刀片切到肌肉。 </li><li>有技术人员2＃进行谈话的主题和按摩对侧下肢。 </li></ol></li><li>有运营商的地方无菌纱布在切口活检部位宽松的数量，并采用直接压到切口，以减少出血。 <ol><li>有技术＃1的3通金属活栓连接到一次性60毫升注射器和30厘米的延长管（ 图1）。 </li><li>有技术人员＃1连接的锥形塑料管连接器的一端插入延伸管的另一端牢牢地插在锥形塑料管连接器的另一端插入200微升枪头的大底与〜15-18毫米前端切掉。 </li></ol></li></ol>.JPG"WIDTH ="500"/&gt; 图1 BERGSTROM针。 （一）伯格斯坦针（5毫米）的是由（A）外插管及（b）内套管针，相关组件包括:（C）柱塞，（D）200微升枪头〜15-18毫米切断，（五）锥形塑料连接器，（F）3通金属塞阀（克）30厘米延伸管，和（h）一次性60毫升注射器（B）的BERGSTROM针（5毫米）和相关的部件组装而成。（ C）的BERGSTROM针（5毫米）的排出的内套管针约1cm开启外部套管的窗口。 <ol start="9"><li>后出血已消退，具有技师＃1打开高压釜灭菌袋以及呈现5毫米活检针和内套管（ 图1）的操作者，同时保持无菌技术。 <ol><li>有运营商组装针（外套管和内套管针），检查路线和滑动的动作。 </li> &lt;/ OL&gt; </li><li>有操作者牢固地保持针头组件（外套管和内套管针），用双手来防止内套管针旋转。有技术人员＃1牢牢插入剪切枪头插入穿刺针的内套管部分的顶部有轻微的向下扭转运动。确保技术人员＃1不接触操作者或活检针，只枪头。 </li><li>有操作者引入活检针插入通过切口的组织。定位在筋膜的切口（后天"感觉"）与所述活检针的尖端。推进针刚刚过去的筋膜，然后角针向下朝向地板，作为将针推入肌肉。注:受试者可能会遇到一些轻微的不适（ 例如 ，"深压力"，或轻度痉挛感觉），为通过筋膜切口位于与所述活检针的尖端并在针被推进到肌肉。 <ol> &lt;李&gt;有技术员＃2指示须留"宽松"，而不是合同的大腿肌肉，而运营商正在推进针头进入肌肉。 </li></ol></li><li>一旦活检针的位置，有运营商的信号技师＃1，打开一次性60毫升注射器。 <ol><li>有技术人员＃1迅速拉一次性60毫升注射器开到40-50毫升标记活检针中创建的吸力。 </li><li>有操作者拉动内套管出大约1cm，同时保持肌肉内外侧套管针的位置，打开外插管的窗口。有运营商迅速关闭内套管剪切（剪辑），并收集肌肉样本。有运营商的信号技师＃1释放吸力。 </li><li>有技术人员＃1打开活塞房间释放吸力。 </li><li>有操作者旋转所述活检针90°并重复上述过程。如果需要的话，有运营商repea吨的旋转操作高达3倍（共4个夹子）。 </li></ol></li><li>有运营商申请直接反压至切口处用无菌纱布，同时去除大腿活检针，小心不要赶筋膜或皮肤。如果显著感觉到阻力，重新切割，同时将扭转运动到内套管针。 </li><li>在除去从大腿的活检针，具有操作者拉动内套管针背1cm至目视检查外套管的内腔中，如果收集组织的足够量来估计。 <ol><li>有操作者手的活检针至技术员＃1。为了保持无菌技术确保技师＃1只顶住针，而不是运营商。有技术人员＃1手活检针来技师＃3。 </li></ol></li><li>具有技师＃3从活检针的组织。 <ol><li>具有技师＃3使用柱塞（ 图1）和一对精尖镊子以确保所有的肌肉组织从内套管和外套管除去。 </li><li>有技师＃3迅速称重样品，以确认被收集肌肉组织的足够量和放置样品的冰冷解剖块上。 </li></ol></li><li>有技术人员3＃仔细解剖可见结缔组织和脂肪的肌肉样本。已技师＃3，根据下游分析制备的肌肉样本进行存储。例如，立即将样品在离心管和捕捉冷冻在液氮中，安装并冻结了组织学，或发生在离心管与RNA酶抑制剂等。 </li><li>如有必要，重复该步骤与第二灭菌活检针，同时保持无菌操作。 </li></ol> 3，封闭<ol><li>有运营商直接施加压力，活检部位用无菌纱布和冰敷10-15分钟。 </li><li>当止血实现，有运营商使用酒精公关EP垫去除干涸的血迹从切口周围的区域。 </li><li>有技术人员＃1目前局部手术胶粘剂涂抹到运营商的管道。有操作者装配手术粘合剂和施加器的管不接触涂抹器头或管的顶端。 </li><li>有操作者拉，用一只手的切口闭合而施加外科粘合剂的单一层以上的干切口用另一只手的顶部。该粘合剂固化（〜90秒）后，将磁带closuresperpendicular的切口。另外，关闭切口用两个4-0的无菌缝合。 </li><li>有操作者施加直接压力通过放置2月3日至4日"×2"非无菌纱布垫的切口部位，并与自粘合粘接缠绕固定。 </li><li>有运营商提供的主体语言和适当的伤口护理，正常和异常反应的书面说明，并为下面的1〜4天的活动准则（在网上看到苏pplement 2）。 </li></ol>

<ol>
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J., 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=Brief+intense+interval+exercise+activates+AMPK+and+p38+MAPK+signaling+and+increases+the+expression+of+PGC-1alpha+in+human+skeletal+muscle.">Brief intense interval exercise activates AMPK and p38 MAPK signaling and increases the expression of PGC-1alpha in human skeletal muscle.</a> J Appl Physiol. 106, 929-934 (1985).</li><li>Phillips, S. M., Tipton, K. D., Ferrando, A. A., Wolfe, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resistance+training+reduces+the+acute+exercise-induced+increase+in+muscle+protein+turnover.">Resistance training reduces the acute exercise-induced increase in muscle protein turnover.</a> Am J Physiol. 276, 118-124 (1999).</li><li>Davis, J. M., Murphy, E. A., Carmichael, M. D., Davis, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quercetin+increases+brain+and+muscle+mitochondrial+biogenesis+and+exercise+tolerance.">Quercetin increases brain and muscle mitochondrial biogenesis and exercise tolerance.</a> Am J Physiol Regul Integr Comp Physiol. 296, 1071-1077 (2009).</li><li>Vissing, K., Andersen, J. L., Schjerling, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Are+exercise-induced+genes+induced+by+exercise.">Are exercise-induced genes induced by exercise.</a> FASEB J. 19, 94-96 (2005).</li><li>Lundby, 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=Gene+expression+in+human+skeletal+muscle:+alternative+normalization+method+and+effect+of+repeated+biopsies.">Gene expression in human skeletal muscle: alternative normalization method and effect of repeated biopsies.</a> Eur J Appl Physiol. 95, 351-360 (2005).</li><li>Coyle, E. F., Coggan, A. R., Hemmert, M. K., Ivy, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+glycogen+utilization+during+prolonged+strenuous+exercise+when+fed+carbohydrate.">Muscle glycogen utilization during prolonged strenuous exercise when fed carbohydrate.</a> J Appl Physiol. 61, 165-172 (1985).</li></ol>

The authors have no conflicts of interest.

这部影片提供了在亚利桑那州立大学人类表现实验室使用的骨骼肌活检过程的步骤一步的总结。此过程中，小的修改，已被用于在过去的13年，收集约1600肌肉活检。肌肉活检样本中的运动营养基础研究提供了重要的数据，从而导致重要的研究发现。 有需要注意进行改性BERGSTROM骨骼肌活检技术时的许多重要的技术。首先，活检队必须在程序的各个阶段保持严格无菌操作。当定?...

经皮或"半开放"活检技术被用来获得从人患者和研究受试者的骨骼肌组织标本。假肥大型（1806年至1875年），相信作为第一个构建针头套管针经皮活检1以获取活体骨骼肌。在20世纪60年代，伯格斯坦推出了经皮活检针类似由杜氏2-4描述。二十年后，Evans 等人的 5通过经由BERGSTROM针的切割套管针施加抽吸改性的技术。这种修饰可以增强组织3的产率，以5倍6,7和被用在临床和生物医学研究的设置。这种技术已经并将继续培养肌病的诊断和我们的骨骼肌的结构和功能的理解。 经皮肌活检技术是straightforward。如果做得正确，严格遵守无菌技术操作，相关的风险是最小的。肌肉活检程序被视为一种研究工具，用于研究项目。一些学术机构允许经过培训的教师与研究人员博士，与医生的直接监督，以获得肌肉活检等人要求委员会认证的医师来执行的技术。在过去的13年8月15日在亚利桑那州立大学活检团队已经成功完成超过1,600肌肉活检。该视频的目的是描述了改性BERGSTROM针经皮肌活检技术5获得骨骼肌组织样品从人类受试者的股外侧。

<table><tbody><tr><td>Bergstr&amp;ouml;m biopsy needle, 5 x 100 mm</td><td>Stille Surgical Inc.</td><td>119-29187-50</td><td>1 ea</td></tr><tr><td>Durasorb blue pad</td><td>Fisher</td><td>22-031-340</td><td>case of 300</td></tr><tr><td>Prep razor disposable</td><td>Moore Medical</td><td>89760</td><td>1 ea</td></tr><tr><td>Shave cream</td><td>Moore Medical</td><td>92869</td><td>1 can</td></tr><tr><td>Alcohol swab</td><td>Moore Medical</td><td>98721</td><td>box/200</td></tr><tr><td>Povidone-iodine swabsticks (3 pack)</td><td>Moore Medical</td><td>90691</td><td>pack of 50</td></tr><tr><td>Face masks</td><td>Fisher</td><td>19-039-690</td><td>case of 50</td></tr><tr><td>Sterile gloves; sz 6.5</td><td>Moore Medical</td><td>68128</td><td>box of 50 pairs</td></tr><tr><td>Sterile gloves; sz 7.0</td><td>Moore Medical</td><td>68129</td><td>box of 50 pairs</td></tr><tr><td>Sterile gloves; sz 8.5</td><td>Moore Medical</td><td>68132</td><td>box of 50 pairs</td></tr><tr><td>Fenestrated towel drape</td><td>Moore Medical</td><td>92712</td><td>case of 50</td></tr><tr><td>Lidocaine (1% HCl, w/o Epi)</td><td>Dealmed</td><td>427902</td><td>30 cc vial</td></tr><tr><td>Ethyl chloride spray</td><td>Dealmed</td><td>386020</td><td>3.5 fl. Oz. bottle</td></tr><tr><td>5 ml syringe w/ 21 G needle</td><td>Fisher</td><td>14-827-48</td><td>case of 100</td></tr><tr><td>25 G 5/8&quot; needle</td><td>Fisher</td><td>14-826AA</td><td>case of 100</td></tr><tr><td>25 G 1 1/2&quot; needle</td><td>Fisher</td><td>14-826-49</td><td>case of 100</td></tr><tr><td>Single-use scalpels w/ #11 blade</td><td>Fisher</td><td>0-8927-5B</td><td>box of 20</td></tr><tr><td>60 ml Syringe</td><td>Fisher</td><td>13-689-8</td><td>case of 40</td></tr><tr><td>~30 inch extension tubing</td><td>Fisher</td><td>14-169-7A</td><td>roll of 50 ft</td></tr><tr><td>Metal stopcock</td><td>Fisher</td><td>01-290-38</td><td>each</td></tr><tr><td>Polypropylene tubing connector w/ tapered ends, 3.2 to 5.5 mm</td><td>Fisher</td><td>15-315-28A</td><td>case of 100</td></tr><tr><td>Yellow pipette tips (1-200 &amp;micro;l)</td><td>Fisher</td><td>02-681-2</td><td>cut off ~15-18 mm of the tip</td></tr><tr><td>Topical surgical adhesive</td><td>Medline</td><td>MSC091076</td><td>case of 36 tubes</td></tr><tr><td>Topical surgical adhesive</td><td>Medline</td><td>MSC091076Z</td><td>box of 6 tubes</td></tr><tr><td>Butterfly stitches (adhesive strips) 1/2&quot; x 4&quot; (6 per pack)</td><td>Moore Medical</td><td>93050</td><td>box/300</td></tr><tr><td>Bandadge, Strip, 3/4 x 3 in.</td><td>Fisher</td><td>19-027-202</td><td>pack of 100</td></tr><tr><td>Adhesive bandage (5 m rolls)</td><td>Fisher</td><td>19-027-768</td><td>case of 36</td></tr><tr><td>1&quot; paper surgical tape</td><td>Fisher</td><td>19-027-761</td><td>case of 12</td></tr><tr><td>Sutures (4-0 reverse cutting, FS2)</td><td>Moore Medical</td><td>82926</td><td>box of 36</td></tr><tr><td>4 x 4 Sterile gauze pads</td><td>Moore Medical</td><td>66949</td><td>pkg/50</td></tr><tr><td>2 x 2 Non-sterile gauze pads</td><td>Moore Medical</td><td>37336</td><td>pkg/200</td></tr><tr><td>3 x 4 Non-adherent pads</td><td>Moore Medical</td><td>74389</td><td>bx/100</td></tr><tr><td>Autoclave bags 3.5 x 9</td><td>Moore Medical</td><td>40390</td><td>box of 200</td></tr><tr><td>Autoclave bags 5.25 x 10</td><td>Moore Medical</td><td>40391</td><td>box of 200</td></tr><tr><td>Autoclave indicator tape</td><td>Moore Medical</td><td>31608</td><td>box of 250</td></tr><tr><td>Lab coats&amp;mdash;small</td><td>Fisher</td><td>23-900-520A&amp;nbsp;</td><td>10/pk</td></tr><tr><td>Lab coats&amp;mdash;medium</td><td>Fisher</td><td>23-900-520B&amp;nbsp;</td><td>10/pk</td></tr><tr><td>Lab coats&amp;mdash;large</td><td>Fisher</td><td>23-900-520C&amp;nbsp;</td><td>10/pk</td></tr><tr><td>Germicidal Disposable Cloth Wipes</td><td>Moore Medical</td><td>62879</td><td>Can/160</td></tr></tbody></table>

human skeletal muscle biopsy procedures using the modified bergstr&#246;m technique

经皮活检技术使研究人员和临床医生收集的骨骼肌肉组织样本。该技术是安全的和高度有效的。这部影片描述了使用修改后的伯格斯坦针，以获得人类受试者的股外侧肌肉组织样本经皮活检技术。该BERGSTROM针包括一个外套管具有在前端侧的小开口（"窗口"）和一个内套管针带切削刃的前端。在局部麻醉和无菌条件下，将针通过皮肤，皮下组织和筋膜切开前进到骨骼肌。接着，抽吸施加到内套管针，所述外套管针被拉回，骨骼肌组织被吸入到所述外套管被抽吸的窗口，并且所述内套管针迅速闭合，从而切断或削波的骨骼肌组织样品。针旋转90°60，另有裁切。这个过程可以重复3次以上。此多刀切割技术通常产生100-200毫克或更多的健康受试者的样品与可以做立即之前，期间和运动或其他介入的回合之后。随着切口部位的后活检敷料，受试者通常恢复日常活动的时候了，并能充分参与身体活动中的48-72小时。受试者应避免重阻力锻炼48小时通过切口在筋膜，减少肌肉症的风险。

如上文所描述的肌肉活检程序允许研究者快速一致收集骨骼肌组织样品。典型的产率在健康，运动受试者是200毫克或更多的单次通过以3-4的剪辑。该过程需要15-20分钟，其中大部分是花在准备在切口。在运动为基础的研究，肌肉样品往往采取前后运动，以恢复过程中收集的一个或两个样本。利用这一设计，在运动后的毛发（对侧）大腿被限幅和切口位点是预先标有一个细点永久性标记，而运动前?...

Watch this Scientific Journal Video about Human Skeletal Muscle Biopsy Procedures Using the Modified BergstrÃ¶m Technique at JoVE.com

Human Skeletal Muscle Biopsy Procedures Using the Modified Bergstr&#246;m Technique

The purpose of this video is to present the modified Bergstr&#246;m skeletal muscle biopsy technique on human subjects.

人骨骼肌活检手术使用修改伯格斯坦技术

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly ...

human-skeletal-muscle-biopsy-procedures-using-modified-bergstrm

Research

JoVE Journal

Medicine

39.8K 次观看.  Appalacian State University. The purpose of this video is to present the modified Bergström skeletal muscle biopsy technique on human subjects. 

视频: Human Skeletal Muscle Biopsy Procedures Using the Modified Bergström Technique

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated from human muscle biopsy samples using enzymatic digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56+ and later as CD56+/desmin+ cells and (ii) muscle-derived fibroblasts, identified as CD56&#8211; and TE-7+. Fibroblasts proliferate very efficiently in culture and in mixed cell populations these cells may overrun myogenic cells to dominate the culture. The isolation and purification of different cell types from human muscle is thus an important methodological consideration when trying to investigate the innate behavior of either cell type in culture. Here we describe a system of sorting based on the gentle enzymatic digestion of cells using collagenase and dispase followed by magnetic activated cell sorting (MACS) which gives both a high purity (&#62;95% myogenic cells) and good yield (~2.8 x 106 &#177; 8.87 x 105 cells/g tissue after 7 days in vitro) for experiments in culture. This approach is based on incubating the mixed muscle-derived cell population with magnetic microbeads beads conjugated to an antibody against CD56 and then passing cells though a magnetic field. CD56+ cells bound to microbeads are retained by the field whereas CD56&#8211; cells pass unimpeded through the column. Cell suspensions from any stage of the sorting process can be plated and cultured. Following a given intervention, cell morphology, and the expression and localization of proteins including nuclear transcription factors can be quantified using immunofluorescent labeling with specific antibodies and an image processing and analysis package.

The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells.

从人类骨骼肌成肌原细胞和成纤维细胞的分离和定量免疫细胞化学表征

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

科研

发育生物学

X-ray Diffraction of Intact Murine Skeletal Muscle as a Tool for Studying the Structural Basis of Muscle Disease

Transgenic mouse models have been important tools for studying the relationship of genotype to phenotype for human diseases including those of skeletal muscle. Mouse skeletal muscle has been shown to produce high quality X-ray diffraction patterns on third generation synchrotron beamlines providing an opportunity to link changes at the level of the genotype to functional phenotypes in health and disease by determining the structural consequences of genetic changes. We present detailed protocols for preparation of specimens, collecting the X-ray patterns and extracting relevant structural parameters from the X-ray patterns that may help guide experimenters wishing to perform such experiments for themselves.

We present detailed protocols for performing small-angle X-ray diffraction experiments using intact mouse skeletal muscles. With the wide availability of transgenic mouse models for human diseases, this experimental platform can form a useful test bed for elucidating the structural basis of genetic muscle diseases

完整毛里丝骨骼肌的X射线衍射作为研究肌肉疾病结构基础的工具

Transgenic mouse models have been important tools for studying the relationship of genotype to phenotype for human diseases including those of skeletal ...

Isolation, Culture, Characterization, and Differentiation of Human Muscle Progenitor Cells from the Skeletal Muscle Biopsy Procedure

The use of primary human tissue and cells is ideal for the investigation of biological and physiological processes such as the skeletal muscle regenerative process. There are recognized challenges to working with human primary adult stem cells, particularly human muscle progenitor cells (hMPCs) derived from skeletal muscle biopsies, including low cell yield from collected tissue and a large degree of donor heterogeneity of growth and death parameters among cultures. While incorporating heterogeneity into experimental design requires a larger sample size to detect significant effects, it also allows us to identify mechanisms that underlie variability in hMPC&#160;expansion capacity, and thus allows us to better understand heterogeneity in skeletal muscle regeneration. Novel mechanisms that distinguish the expansion capacity of cultures have the potential to lead to the development of therapies to improve skeletal muscle regeneration.

We present techniques for isolating, culturing, characterizing, and differentiating human primary muscle progenitor cells (hMPCs) obtained from skeletal muscle biopsy tissue. hMPCs obtained and characterized through these methods can be used to subsequently address research questions related to human myogenesis and skeletal muscle regeneration.

从骨骼肌活检程序分离、培养、表征和分化人类肌肉祖细胞

The use of primary human tissue and cells is ideal for the investigation of biological and physiological processes such as the skeletal muscle ...

Performing Human Skeletal Muscle Xenografts in Immunodeficient Mice

Treatment effects observed in animal studies often fail to be recapitulated in clinical trials. While this problem is multifaceted, one reason for this failure is the use of inadequate laboratory models. It is challenging to model complex human diseases in traditional laboratory organisms, but this issue can be circumvented through the study of human xenografts. The surgical method we describe here allows for the creation of human skeletal muscle xenografts, which can be used to model muscle disease and to carry out preclinical therapeutic testing. Under an Institutional Review Board (IRB)-approved protocol, skeletal muscle specimens are acquired from patients and then transplanted into NOD-Rag1null IL2r&gamma;null (NRG) host mice. These mice are ideal hosts for transplantation studies due to their inability to make mature lymphocytes and are thus unable to develop cell-mediated and humoral adaptive immune responses. Host mice are anaesthetized with isoflurane, and the mouse tibialis anterior and extensor digitorum longus muscles are removed. A piece of human muscle is then placed in the empty tibial compartment and sutured to the proximal and distal tendons of the peroneus longus muscle. The xenografted muscle is spontaneously vascularized and innervated by the mouse host, resulting in robustly regenerated human muscle that can serve as a model for preclinical studies.

Complex human diseases can be challenging to model in traditional laboratory model systems. Here, we describe a surgical approach to model human muscle disease through the transplantation of human skeletal muscle biopsies into immunodeficient mice.

在免疫缺陷小鼠中表演人类骨骼肌异种移植物

Treatment effects observed in animal studies often fail to be recapitulated in clinical trials. While this problem is multifaceted, one reason for this ...

Quantification of Subcellular Glycogen Distribution in Skeletal Muscle Fibers using Transmission Electron Microscopy

With the use of transmission electron microscopy, high-resolution images of fixed samples containing individual muscle fibers can be obtained. This enables quantifications of ultrastructural aspects such as volume fractions, surface area to volume ratios, morphometry, and physical contact sites of different subcellular structures. In the 1970s, a protocol for enhanced staining of glycogen in cells was developed and paved the way for a string of studies on the subcellular localization of glycogen and glycogen particle size using transmission electron microscopy. While most analyses interpret glycogen as if it is homogeneously distributed within the muscle fibers, providing only a single value (e.g., an average concentration), transmission electron microscopy has revealed that glycogen is stored as discrete glycogen particles located in distinct subcellular compartments. Here, the step-by-step protocol from tissue collection to the quantitative determination of the volume fraction and particle diameter of glycogen in the distinct subcellular compartments of individual skeletal muscle fibers is described. Considerations on how to 1) collect and stain tissue specimens, 2) perform image analyses and data handling, 3) evaluate the precision of estimates, 4) discriminate between muscle fiber types, and 5) methodological pitfalls and limitations are included.

A modified post-fixation procedure increases the contrast of glycogen particles in tissue. This paper provides a step-by-step protocol describing how to handle the tissue, conduct the imaging, and use stereological methods to obtain unbiased and quantitative data on fiber type-specific subcellular glycogen distribution in skeletal muscle.

使用透射电子显微镜定量骨骼肌纤维中的亚细胞糖原分布

With the use of transmission electron microscopy, high-resolution images of fixed samples containing individual muscle fibers can be obtained. This ...

生物化学

Preparation and Respirometric Assessment of Mitochondria Isolated from Skeletal Muscle Tissue Obtained by Percutaneous Needle Biopsy

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining samples of human Vastus lateralis, isolating mitochondria from minimal amounts of skeletal muscle tissue, and plate based respirometric profiling using an extracellular flux (XF) analyzer. Comparison of respirometric profiles obtained using 1.0, 2.5 and 5.0 &#956;g of mitochondria indicate that 1.0 &#956;g is sufficient to measure respiration and that 5.0 &#956;g provides most consistent results based on comparison of standard errors. Western blot analysis of isolated mitochondria for mitochondrial marker COX IV and non-mitochondrial tissue marker GAPDH indicate that there is limited non-mitochondrial contamination using this protocol. The ability to study mitochondrial respirometry in as little as 20 mg of muscle tissue allows users to utilize individual biopsies for multiple study endpoints in clinical research projects.

Methods for biopsy of Vastus lateralis, preparation of purified mitochondria, and respirometric profiling are described. The use of small muscle volume makes this technique suitable for clinical research applications.

线粒体的制备及透气性评估，从骨骼肌组织通过穿刺活检获得的分离

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining ...

医学

MyDoBID- 一种有效识别肌肉纤维类型的新技术

Fiber Type Identification of Human Skeletal Muscle

The technique described here can be used to identify specific myosin heavy chain (MHC) isoforms in segments of individual muscle fibers using dot blotting, hereafter referred to as Myosin heavy chain detection by Dot Blotting for IDentification of muscle fiber type (MyDoBID). This protocol describes the process of freeze-drying human skeletal muscle and isolating segments of single muscle fibers. Using MyDoBID, type I and II fibers are classified with MHCI- and IIa-specific antibodies, respectively. Classified fibers are then combined into fiber type-specific samples for each biopsy.
The total protein in each sample is determined by Sodium Dodecyl-Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) and UV-activated gel technology. The fiber type of samples is validated using western blotting. The importance of performing protein loading normalization to enhance target protein detection across multiple western blots is also described. The benefits of consolidating classified fibers into fiber type-specific samples compared to single-fiber western blots, include sample versatility, increased sample throughput, shorter time investment, and cost-saving measures, all while retaining valuable fiber type-specific information that is frequently overlooked using homogenized muscle samples. The purpose of the protocol is to achieve accurate and efficient identification of type I and type II fibers isolated from freeze-dried human skeletal muscle samples.
These individual fibers are subsequently combined to create type I and type II fiber type-specific samples. Furthermore, the protocol is extended to include the identification of type IIx fibers, using Actin as a marker for fibers that were negative for MHCI and MHCIIa, which are confirmed as IIx fibers by western blotting. Each fiber type-specific sample is then used to quantify the expression of various target proteins using western blotting techniques.

作者聚焦:使用 MyDoBID 技术破译骨骼肌纤维类型的奥秘 

This protocol demonstrates single-fiber isolation from freeze-dried human skeletal muscle and fiber-type classification according to Myosin heavy chain (MHC) isoform using the dot blotting technique. Identified MHC I and II fiber samples can then be further analyzed for fiber type-specific differences in protein expression using western blotting.

人体骨骼肌的纤维类型鉴定

The technique described here can be used to identify specific myosin heavy chain (MHC) isoforms in segments of individual muscle fibers using dot ...

Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome

Percutaneous muscle biopsy using the Weil-Blakesley conchotome is well established in both clinical and research practice. It is a safe, effective and well tolerated technique. The Weil-Blakesley conchotome has a sharp biting tip with a 4 - 6 mm wide hollow. It is inserted through a 5 - 10 mm skin incision and can be maneuvered for controlled tissue penetration. The tip is opened and closed within the tissue and then rotated through 90 -180&#176; to cut the muscle. The amount of muscle obtained following repeated sampling can vary from 20 mg to 290 mg which can be processed for both histology and molecular studies. The wound needs to be kept dry and vigorous physical activity kept to a minimum for approximately 72 hr although normal levels of activity can restart immediately following the procedure. This procedure is safe and effective when close attention is paid to the selection of subjects, full asepsis and post procedure care. &#160;Both right and left vastus lateralis are suitable for biopsy dependent on participant preference.

Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome

This video demonstrates the technique of percutaneous muscle biopsy of the human vastus lateralis using the Weil-Blakesley conchotome.

人的<em&gt;股外侧肌</em&gt;骨骼肌活检使用韦尔 - Blakesley Conchotome

Percutaneous muscle biopsy using the Weil-Blakesley conchotome is well established in both clinical and research practice. It is a safe, effective and ...

人骨骼肌活检手术使用修改伯格斯坦技术

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