The Method of Primary Cell Culture of Rat's Gastrocnemius Muscle and Comparison between Skeletal Muscle Cell and Tendon Cell in Protein Expressions of Myogenic Factor 5, Paired Box 7, and Desmin

大鼠腓腸肌細胞原代培養方法及生肌因數5（Myogenic Factor 5）、配對盒基因7（Paired Box 7）和肌間線蛋白（Desmin）在骨骼肌細胞與肌腱細胞上的蛋白質表現

10.6315/2014.42(1)03

陳盈勳(Ying-Hsun Chen)；蔡文鐘(Wen-Chung Tsai)；林妙穗(Miao-Sui Lin)；蘇中慧(Jong-Hwei S. Pang)；潘冠誠(Kuan-Chen Pan)；游東陽(Tung-Yang Yu)

骨骼肌細胞 ； 生肌因數5 ； 配對盒基因7 ； 肌間線蛋白 ； 原代培養 ； skeletal muscle cell ； Myf5 ； Pax7 ； desmin ； primary culture

台灣復健醫學雜誌

42卷1期（2014 / 03 / 01）

23 - 29

英文

骨骼肌最主要的功能是讓肢體產生力量並使關節運動。當骨骼肌受損時，骨骼肌細胞的修復會開始進行。因此，了解骨骼肌細胞的生物、生理、及病理機轉有助於調控骨骼肌細胞修復。為了要進行骨骼肌細胞的相關研究，我們需要培養大量的骨骼肌細胞。目前最常用的方式是利用細胞株培養來產骨骼肌細胞。藉由細胞株培養的優點是能產生大量的細胞。然而，這些培養出的細胞的部分特性跟原來的細胞已有些許不同。原代培養所產生細胞的主要特性與原來的細胞較類似。經由原代培養所產生的細胞適合用於骨骼肌損傷及修補等相關研究。我們的研究目的是用大鼠腓腸肌細胞來建立一個骨骼肌細胞的原代培養的流程。一開始先取出大鼠腓腸肌細胞放在離心管內，然後利用酵素將細胞切割開。經過離心後，將上清液放置在特殊的培養基上。第一個附著在培養基上的是纖維母細胞。將未附著的細胞放到另一培養基培養。經過二十四小時培養後，將附著於培養基上的細胞用磷酸類溶液沖洗後再培養即得所要的細胞。藉由這個流程所培養出的細胞經過西方點墨法分析後，發現有生肌因數5、配對盒基因7 及肌間線蛋白所產生的蛋白質。這三個蛋白質只會出現在骨骼肌細胞。所以，我們可以藉由這三個蛋白質的產生，確認所培養出的細胞為骨骼肌細胞。

Skeletal muscle cells generate force and produce joint movement and are easily injured. A good understanding of the biological, physiological, and pathological mechanisms of skeletal muscle cells is essential for exploring the regulation of muscle repair, which occurs in the event of an injury. A method for reproducing skeletal muscle cells is essential for studying skeletal muscles. Most studies use cell lines as study models because these are capable of reproducing numerous times. However, the function of cell lines is markedly deviated from that of normal cells. Skeletal muscle cells from primary cell culture more closely represent muscle cells in vivo. They are better sources for the further study of the skeletal muscles, including the evaluation of the mechanisms underlying muscle damage and repair. In the present study, we used the gastrocnemius muscle from Sprague Dawley rats to establish a protocol for primary skeletal muscle cell culture in vitro. Initially, the gastrocnemius muscle was excised from the rats and placed in a centrifuge tube. We used enzymes to separate the cells. After centrifugation, the supernatant was collected on a special medium. The first adherent cells on the culture plate were fibroblasts. The non-adherent cells were collected for further culture. After 24 hours of incubation, the adherent cells were washed with phosphate-buffered saline and kept cultured in the medium until confluence. These cells then underwent western blot analysis. The results revealed expressions of myogenic factor 5 (Myf5), paired box 7 (Pax7), and desmin proteins, which are only usually present in skeletal muscles. These findings provided evidence of the presence of skeletal muscle cells in the culture.