- 期刊
肌肉穿刺技術在運動科學研究的應用
Muscle Biopsy Technique in Exercise Science Research
摘要
人類骨骼肌在維持生命品質中扮演著至關重要的角色，早期的動物研究已經證實，骨骼肌在應對各種損傷挑戰時具有非凡的修復和再生能力。這些發現促使肌肉穿刺（muscle biopsy）技術被引入人體研究中，這一技術極大地推動了人類如何適應身體挑戰，以及從運動中獲得健康益處的理解。截至目前，已有超過10,000篇經過同儕評審的文章發表這一侵入性技術。肌肉穿刺在揭示骨骼肌如何通過改變基因表達來適應壓力、骨髓來源的細胞如何促進肌肉組織的更新、能量基質在運動期間和運動後的利用方式等方面發揮了關鍵作用，推動運動科學的知識進展。1965年，Jonas Bergström設計了首支運動科學研究之肌肉穿刺針，他的初期研究成果陸續於1966年和1967年發表在Acta Medica Scandinavica和Acta Physiologica Scandinavica期刊上（Ahlborg et al., 1967; Bergström & Hultman, 1966）。他們招募了9名健康的參與者，在進行75% VO_(2max)的肌肉肝糖耗竭運動挑戰前進行肌肉穿刺。隨後，參與者在交叉實驗設計下食用了熱量相等的三種飲食：混合飲食、脂肪－蛋白質飲食及高碳水化合物的飲食。Bergström發現，因肌肉肝醣含量較高，高碳水化合物飲食後的耐力時間顯著較長（189分鐘），相比之下，脂肪－蛋白質飲食（59分鐘）和混合飲食（126分鐘）則較短。這項突破性發現奠定了碳水化合物作為人類耐力運動的主要營養素的重要性。目前肌肉穿刺已應用在臨床醫學上，作為診斷肌肉疾病的重要技術，例如：肌肉營養不良症、多發性肌炎和肌原纖維肌病等，通過取得人體肌肉樣本的醫療診斷結果，提升醫師在神經系統、結締組織、血管系統及骨骼肌肉系統疾病判讀的準確性。從肉組織取得肌肉樣本的程序包括閉鎖式（needle biopsy）和開放式（open biopsy）兩種肌肉穿刺技術。閉鎖式穿刺使用細長針插入肌肉組織，抽取一小塊組織樣本；開放式穿刺則通過小切口直接從肌肉中取出較大塊組織樣本（Ray-Coquard et al., 2003）。粗針穿刺（core needle biopsy）使用一種特殊的空心針，通過皮膚和肌肉層取得較大的組織柱狀樣本（Bruening et al., 2010）；真空輔助穿刺（vacuum-assisted biopsy）結合真空技術，通過一個較大的針孔可反覆抽取多個組織樣本（Skrzynski et al., 1996）。基於醫療檢查需求，這些技術有不同的使用需求及其各自的優缺點（表1）（Kasraeian et al., 2010）。（表格略）如何提升肌肉穿刺技術以取得更多組織樣本量，Evans et al.（1982）提出改良的肌肉穿刺取樣（真空輔助穿刺），並在Medicine & Science in Sports & Exercise期刊上發表了一種增加骨骼肌破皮穿刺樣本取樣方法的描述，在肌肉穿刺針穿透皮膚並插入受試者的肌肉後，通過抽吸針內的空氣壓力來促進肌肉組織嵌入針孔內的凹處，經過彈簧切片後可以獲得更大的肌肉組織量（約78.5mg）。肌肉採樣位置大部分（約92%）選擇人體大腿股外側肌群（vastus lateralis muscle group）（Newmire & Willoughby, 2022）。Chen et al.（2019）指出，如何避免受試者因肌肉穿刺而引起的不良事件是非常需要考慮的重點。Chen et al.指出，大腿股外側肌群是一個肌肉量豐富且易於取樣的部位，且其神經和血管分布較少，這降低了取樣過程中出血和神經損傷的風險。人體股外側肌群神經血管分支最少的區域位於外側筋膜（骼束）的前面，距側面25%～50%之間至大轉子的關節線。大腿股外側肌群也是常見運動中站立姿勢中使用的肌肉之一，可能是它能有效代表整個身體的運動能力。這些生理解剖和運動知識的原因使得大腿股外側肌部位成為肌肉活組織切片採集的首選位置（Chen et al., 2019）。當然，也有少數因不同研究目的進行的人體肌肉取樣，例如：腓腸肌（gastrocnemius）、比目魚肌（soleus）、背闊肌（latissimus dorsi）、肱三頭肌（triceps brachii）、腰多裂肌（lumbar multifidus）、豎脊肌（erector spinae）和斜方肌（upper trapezius），通常會使用利多卡因（lidocaine）局部麻醉劑，減少肌肉穿刺採集樣本過程中的不適感。本文實驗受試者大腿股外側肌肉上皮局部注射2%利多卡因，使用量為1～2毫升。自1965年Bergström及1982年Evans et al.提出肌肉穿刺技術在運動科學研究後，隨後發表期刊文章顯示該技術可應用於運動科學領域涵蓋了運動肌肉生理、運動表現、阻力型運動、有氧運動訓練、運動肌肉傷害及運動肌肉肝醣、粒線體生合成等多個研究議題（圖1）。Iaia et al.（2011）探討人體不同運動強度表現與骨骼肌細胞特徵關係。7名運動選手挑戰8～10次高強度腳踏車運動（運動負荷：150～700 W，運動時間20秒至150分鐘）。Iaia et al.指出，30秒內的高強度運動表現與肌肉纖維快縮肌比例高度相關，而肌肉細胞的微血管密度和鈉－鉀幫浦在30秒至4分鐘的高強度腳踏車運動中扮演著極其重要的角色。另外，肌肉穿刺技術在運動訓練方式的應用探討，例如血流限制（blood flow restriction, BFR）訓練對運動訓練生理效果的研究。Christiansen et al.（2019）招募12名受試者進行BFR腳踏車6週的運動訓練。BFR提升血管通透性、氧化壓力、引起運動生理適應（Bennett & Slattery, 2019; Christiansen et al., 2019）。人體肌肉樣本可以透過PAS染色法（periodic acid-Schiff stain）分析肌肉肝醣濃度，以及反轉錄聚合酶連鎖反應（reverse transcription polymerase chain reaction, RT-PCR）、西方墨點法（western-blotting）和免疫組織化學（immunohistochemistry, IHC）來瞭解肌肉細胞在分子層次上的變化。近年來與實驗夥伴醫師合作有關運動科學人體肌肉穿刺相關研究成果有運動營養品在運動競技增能劑使用的可行性，以及肌肉肝醣回補、肌肉粒線體生合成的影響（Cheng et al., 2024; Huang et al., 2020）。近幾年研究發現年輕成人肌肉中存在明顯數量的衰老幹細胞且中等強度有氧運動誘發的發炎程度不足以清除人體衰老細胞（Wu et al., 2019），這是當前肌肉生理知識的一項重要結果。臺灣將肌肉穿刺技術應用在運動科學領域仍有發展的空間，雖然肌肉穿刺技術方法經歷了多次修正。然而，仍然對於人體實驗受試者有極大生理或心理壓力。為克服這個問題，本文實驗採用14號肌肉穿刺針的技術步驟（圖2），實驗受試者大腿股外側肌肉上皮局部注射2%利多卡因，使用量為1～2毫升。相較於其他肌肉穿刺方法，例如：開放式穿刺、粗針穿刺和真空輔助穿刺，其優點在於創傷小、恢復快且提升受試者接受度。透過持續改善肌肉穿刺技術，本文希望提供臺灣運動科學研究者運動訓練、補充劑及健康策略的研究工具選項。
關鍵字
無資料並列摘要
Human skeletal muscle plays a vital role in maintaining quality of life by enabling mobility that facilitates experiencing the world. Early animal studies demonstrated the extraordinary reparative and regenerative capabilities of skeletal muscle in response to various damaging challenges. These findings prompted the introduction of muscle biopsy techniques in human exercise science research. This approach has significantly advanced our understanding of how human muscles adapt to physical challenges and derive health benefits from exercise. To date, over 10,000 peer-reviewed articles have been published using this invasive technique. Muscle biopsies have been instrumental in uncovering insights into how muscles alter gene expression to adapt to stress, how bone marrow-derived cells contribute to muscle tissue renewal, and how energy substrates are utilized during and after exercise. This has significantly advanced our knowledge in exercise science. In 1965, Jonas Bergström designed the first muscle biopsy needle and applied it to exercise science research. His initial studies were published in Acta Medica Scandinavica and Acta Physiologica Scandinavica in 1966 and 1967 (Ahlborg et al., 1967; Bergström & Hultman, 1966). He recruited nine healthy participants to undergo muscle biopsies before completing a glycogen-depleting exercise at 75% VO_(2max). Following this, participants consumed one of three diets with equal caloric content: a mixed diet, a fat ± protein diet, or a carbohydrate-rich diet, in a crossover design. Bergström discovered that endurance time was significantly higher (189 minutes) following the high-carbohydrate diet, due to greater muscle glycogen content, compared to the fat-protein diet (59 minutes) and the mixed diet (126 minutes). This breakthrough established the critical role of carbohydrates as the primary macronutrient for human endurance performance. Evans et al. (1982) subsequently proposed an improved muscle biopsy technique, published in the Medicine & Science in Sports & Exercise. This method recommends the use of vacuum-assisted biopsy to obtain larger muscle samples. Medical diagnostic results from human muscle tissue samples enhance doctors' precision in diagnosing diseases of the nervous system, connective tissue, vascular system, and musculoskeletal system. In clinical medicine, muscle biopsy involving needle biopsy and punch biopsy to obtain muscle tissue is a vital technique for diagnosing various muscle diseases such as muscular dystrophy, polymyositis, and myofibrillar myopathy in humans. Needle biopsy involves inserting a thin needle into the muscle to extract a small tissue sample. Open biopsy requires making a small incision to directly remove a larger piece of muscle tissue (Ray-Coquard et al., 2003). Core needle biopsy utilizes a special hollow needle to obtain a larger cylindrical tissue sample through the skin and muscle layers (Bruening et al., 2010). Vacuum-assisted biopsy combines vacuum technique with a larger needle to repeatedly extract multiple tissue samples through a single puncture (Skrzynski et al., 1996). There will be varying usage requirements depending on medical examination needs, along with their respective advantages and disadvantages, as shown in Table 1 (Kasraeian et al., 2010). Local anesthetics like lidocaine are usually administered to minimize discomfort during the procedure. In our laboratory, 2% lidocaine is typically used at the biopsy site, with doses ranging from 1 ~ 2 ml depending on the participant's needs to ensure effective anesthesia. Muscle biopsy samples allow detailed analysis of muscle structure and function through methods like periodic acid-Schiff (PAS) stain for glycogen concentration, reverse transcription polymerase chain reaction (RT-PCR), Western blotting, and immunohistochemistry. In recent years, our team has collaborated with a physician to explore ergogenic aids in athletic competitions, including nutritional approaches with varied glycemic indexes for improved glucose uptake, effects on muscle glycogen replenishment, and impacts on mitochondrial biogenesis (Cheng et al., 2024; Huang et al., 2020). In aging research, muscle biopsy technique has revealed a high number of aging stem cells in the muscles of young adults and demonstrated that moderate-intensity aerobic exercise alone may not adequately clear senescent cells (Wu et al., 2019). These findings represent significant advances in our understanding of muscle physiology. Muscle biopsy technique, initiated by Bergström in 1965 and later improved by Evans et al. in 1982, has also been widely used in exercise and sports science. Applications include studies on muscle physiology, sports performance, exercise-induced muscle damage, muscle energy substrates during exercise, and mitochondrial biogenesis (Figure 1). For example, Iaia et al. (2011) demonstrated that high-intensity exercise performance is associated with fast-twitch muscle fibers, while muscle capillaries and the Na+-K+ pump β1-subunit influence performance in shorter, intense activities. In addition, muscle biopsy has been utilized to study alternative exercise training methods, such as blood flow restriction (BFR) training. Christiansen et al. (2019) conducted a six-week BFR training study that revealed significant changes in muscle glucose utilization and antioxidant enzyme activity in the thigh muscles. Other researchers have found that BFR training increases muscle vascular permeability and free radical production, leading to physiological adaptations (Bennett & Slattery, 2019; Christiansen et al., 2019). Most studies in exercise and sports sciences that use biopsy protocols (about 92%) focus on the vastus lateralis muscle group (Newmire & Willoughby, 2022). The vastus lateralis muscle is commonly chosen because it has a significant muscle mass, is easily accessible, and has a low density of neurovascular branches, which minimizes the risk of complications. Moreover, the vastus lateralis is actively engaged in typical sports stances, making it an ideal muscle for studies aiming to represent whole-body movement. These anatomical and functional considerations support its use for muscle biopsy (Chen et al., 2019). Emphasizing the importance of preventing adverse events during muscle biopsy procedures, Chen et al. (2019) identified the safest biopsy site as the area just anterior to the lateral fascia (iliotibial band), between 25% ~ 50% of the distance from the lateral joint line to the greater trochanter. Some studies also target other muscles, such as the gastrocnemius, soleus, latissimus dorsi, triceps brachii, lumbar multifidus, erector spinae, obliques, and upper trapezius. (The form abridges) In Taiwan, the adoption of muscle biopsy technique has expanded, offering a valuable research tool for exercise science. Although muscle biopsy methods have evolved to reduce invasiveness, psychological and physical stress for participants remains a concern. To address this, we have adopted a modified biopsy approach using two 14-gauge needles to increase sample size while minimizing trauma (Figure 2). This technique improves sample collection efficiency and participant comfort, making it more feasible for human studies compared to traditional methods like open biopsy, core needle biopsy, and vacuum-assisted biopsy. By continuing to develop and refine muscle biopsy methods, we aim to support sports science research in Taiwan and facilitate studies on exercise training, supplements, and health strategies.