一种新型的甲状肌纤维培养方法。
A Novel Method for Thyroarytenoid Myofiber Culture.
机构信息
Rehabilitation Medicine, NYU Grossman School of Medicine, New York City, New York, USA.
Otolaryngology-Head and Neck Surgery, NYU Grossman School of Medicine, New York City, New York, USA.
出版信息
Laryngoscope. 2023 Nov;133(11):3109-3115. doi: 10.1002/lary.30756. Epub 2023 May 25.
OBJECTIVES/HYPOTHESIS: Myofiber culture has been employed to investigate muscle physiology in vitro and is well-established in the rodent hind limb. Thyroarytenoid (TA) myofiber culture has not been described, providing an opportunity to employ this method to investigate distinct TA myofiber functions. The purpose of this study was to assess the feasibility of a TA myofiber culture model.
STUDY DESIGN
In vitro.
METHODS
TA muscles from five Sprague Dawley rats were independently isolated and digested for 90 min. A smooth-tip, wide-bored pipette dissociated TA myofibers from cartilage, and the fibers were distributed on collagen-coated dishes and incubated at 37°C, 5% CO for 2 h. Myofiber specificity was determined via immunolabeling for desmin and myosin heavy chain (MHC). Myofibers viability was assessed over 7 days via esterase assay. Additional myofibers were immunolabeled for satellite cell marker Pax-7. Glucocorticoid (GC) receptor (GR) was immunolabeled following GC treatment.
RESULTS
The harvest technique yielded ~120 myofibers per larynx. By day 7, ~60% of the fibers remained attached and were calcein AM-positive/ethidium homodimer-negative, indicating viability. Myofibers were positive for desmin and MHC, indicating muscle specificity. Cells surrounding myofibers were positive for Pax-7, indicating the presence of myogenic satellite cells. Myofibers also responded to GC treatment as determined by GR nuclear translocation.
CONCLUSION
TA myofibers remained viable in culture for at least 7 days with a predictable response to exogenous stimuli. This technique provides novel investigative opportunities regarding TA structure and function.
LEVEL OF EVIDENCE
N/A Laryngoscope, 133:3109-3115, 2023.
目的/假设：肌纤维培养已被用于研究体外肌肉生理学，并且在啮齿动物后肢中得到了很好的建立。甲状腺上肌（TA）肌纤维培养尚未被描述，这为采用这种方法研究不同 TA 肌纤维功能提供了机会。本研究旨在评估 TA 肌纤维培养模型的可行性。
研究设计
体外。
方法
将五只 Sprague Dawley 大鼠的 TA 肌肉分别独立分离并消化 90 分钟。使用尖端光滑、宽孔的吸管从软骨中分离 TA 肌纤维，然后将纤维分布在涂有胶原蛋白的培养皿上，并在 37°C、5%CO2 下孵育 2 小时。通过免疫标记肌联蛋白和肌球蛋白重链（MHC）来确定肌纤维的特异性。通过酯酶测定在 7 天内评估肌纤维的活力。对其他肌纤维进行卫星细胞标志物 Pax-7 的免疫标记。在给予糖皮质激素（GC）处理后，对 GC 受体（GR）进行免疫标记。
结果
收获技术从每个喉中获得约 120 条肌纤维。在第 7 天，约 60%的纤维仍然附着，并且钙黄绿素 AM 阳性/乙锭同型二聚体阴性，表明活力。肌纤维对肌联蛋白和 MHC 呈阳性，表明肌肉特异性。肌纤维周围的细胞对 Pax-7 呈阳性，表明存在肌源性卫星细胞。肌纤维对 GC 处理的反应也通过 GR 核易位来确定。
结论
TA 肌纤维在培养中至少存活 7 天，对外源刺激有可预测的反应。这项技术为研究 TA 的结构和功能提供了新的研究机会。
证据水平
无。喉镜，133:3109-3115, 2023.
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