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单一肌纤维培养测定法用于评估体外成人肌肉干细胞功能
Chapters
Summary February 15th, 2021
Please note that all translations are automatically generated.
Click here for the English version.
在该协议中，描述了肌肉干细胞的体外培养和功能分析方法，该方法保留了它们与其内源性生态位的大部分相互作用。
Transcript
肌肉干细胞仍然与其内源性生态位相关，并且可以很容易地纵，例如，通过 siRNA 转染。这种方法使得通过保留生态位，可以在比标准2D培养模型更接近体内情况的环境中分析培养物中的肌肉干细胞。首先用钻石笔切割无菌的巴氏移液器。
对于每只小鼠，使用一个开口约0.3厘米，长度约10至12厘米的大口径移液器和第二个玻璃移液器，其小开口约0.1厘米，长度约为22厘米。通过轻柔移动将移液器吸头放在本生燃烧器的火焰中5至10秒，使两个移液器的边缘变得平滑。使用前，立即用无菌马血清涂覆两个移液器，在整个移液器中加入2毫升马血清5分钟，然后注射马血清并让移液管在室温下干燥五分钟。
用70%乙醇喷洒所有设备和小鼠的后肢。使用硬化的细弯曲剪刀和细镊子去除皮肤并暴露下面的肌肉。用细细弯曲的镊子去除周围的筋膜，而不会损坏下面的肌肉。
要切除胫骨前部或TA，请用镊子抓住远端TA肌腱并用细剪刀切割。将TA保持在肌腱处时，将其拉向膝盖并切开靠近膝盖的肌肉以暴露EDL肌肉。用弯曲的镊子抬起远端EDL肌腱，并用精细的Vannas弹簧剪刀切割。
通过小心地将EDL拉向膝盖来暴露近端EDL肌腱。然后用剪刀剪断近端肌腱。在循环水浴中以37摄氏度将EDL肌肉在反应管中孵育。
当肌肉松弛并且可以看到单英里纤维时，停止消化。在装有加热板的无菌双目显微镜下工作，使用大口径移液器用温隔离介质冲洗肌肉。用大口径移液器解离肌肉，直到所需数量的肌纤维在溶液中自由漂浮。
要清洗碎屑，请使用小口径玻璃移液器将非收缩的肌纤维转移到充满隔离介质的第二孔中。然后将50至100个非收缩肌纤维转移到充满肌纤维培养基的24孔板中的一个孔中。将肌纤维在37摄氏度和5%二氧化碳下孵育72至96小时。
在肌纤维分离后四小时转染肌纤维相关的肌肉干细胞。将25微升Opti-MEM与相应体积的siRNA与含有1.5微升转染试剂的25微升Opti-MEM混合。孵育反应混合物五分钟，并将其加入24孔板中的细胞中。
然后将板在37摄氏度下孵育，以示时间的尊重。这里仅演示了免疫荧光染色方案的关键步骤。小心地丢弃肌纤维培养基，同时在孔中留下一些溶液。
加入500微升2%多聚甲醛，以固定肌纤维及其相邻的肌肉干细胞。将板在室温下孵育五分钟。小心地取出上清液，并用PBS洗涤肌纤维三次。
在用二抗孵育后的孵育步骤中用锡箔纸覆盖板。使用疏水笔在微观载玻片上画一个圆圈。然后将尽可能小体积的肌纤维转移到载玻片上并分散它们。
用小口径巴氏移液器或200微升移液器去除残留液体。使用两滴水性安装介质，并用盖玻片覆盖肌纤维。然后让载玻片干燥，并在黑暗中以四摄氏度的温度储存，然后进行显微镜分析。
该协议证明了单肌纤维从小鼠EDL肌肉的衍生和培养。Pax7的免疫荧光染色用于鉴定肌肉干细胞的细胞核。放大区域暴露肌纤维及其相邻的肌肉干细胞，并在肌肉干细胞的细胞核中显示PAX7免疫荧光信号。
肌肉干细胞的肌原进展可以通过标记物表达来分析。Pax7的存在和MyoD表达的缺失与新鲜分离的肌纤维的静止肌肉干细胞有关。MyoD表达可以在增殖的肌肉干细胞中观察到。
72小时后，肌肉干细胞形成具有不同肌原状态的后代簇，这与不同肌原标志物的表达平行。只有Pax7细胞是自我更新的干细胞。Pax7和MyoD双阳性细胞正在增殖。
而只有肌D细胞是分化细胞。肌肉干细胞的siRNA转染显示细胞质siRNA以颗粒方式积累，表明有效摄取。对每个肌纤维转染的Pax7阳性细胞的定量显示，转染细胞的数量在30小时后增加到74%，对肌肉干细胞数量没有不利影响。
在尝试此协议时，仔细剖析EDL而不造成任何损害至关重要。除了siRNA转染外，转基因动物的分析或与重组蛋白的孵育对于相邻肌纤维上肌肉干细胞的进一步功能分析也是可能的。
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