골격근 내 운동 효과검증을 위한 웨스턴블랏에 적합한 loading control
The best loading control in western blot for the verification of adaptive responses of exercise in skeletal muscle.
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초록
본 연구의 목적은 골격근 내 운동의 효과를 검증하기 위한 방법으로서 웨스턴블랏을 실시할 경우 어떠한 loading control을 사용하는 것이 가장 적절한지 알아봄으로서 추후 이와 관련된 연구의 올바른 결과해석을 위한 도움을 주는 것이다. 이를 위해 1일, 3일 그리고 3주간 하루 6시간씩 수영운동을 실시한 후 국내 운동과학 분야에서 주로 사용되는 loading control(actin, β-actin, GAPDH, β-tubulin)의 골격근 내 발현양상을 분석하였다. 그 결과 loading control을 사용하지 않은 연구가 약 40%로 가장 많았으며, 그 다음으로 pan-actin, β-actin, GAPDH, β-tubulin 순이었다. pan-actin과 β-tubulin의 발현은 장기간 운동 후 증가하였으며, GAPDH의 발현은 단기간 운동 후 감소하였다. 그리고 β-actin의 발현은 운동기간과는 상관없이 일정하였다. 따라서 pan-actin과 β-tubulin은 단기간, 그리고 GAPDH는 장기간 운동에 따른 변화양상을 분석할 때 이용이 가능할 것이나, 운동에 따른 골격근 내 변화양상이 거의 없는 β-actin을 사용하여 목표 단백질을 표준화 하는 것이 가장 바람직 할 것이다.
Abstract
The purpose of this study is to show which loading control is the best to use in western blots, in order to correctly analyze and interpret skeletal muscle adaptive responses of exercise. Articles in the Korean exercise science field within the past decade were searched to determine four most fequently used loading controls. These loading controls (actin, β-actin, GAPDH, β-tubulin) were tested for stability during acute and chronic endurance exercise training. After that twenty-four rats were randomly assigned into 4 groups (sedentary, 1 day exercise, 3 day exercise, 3 weeks exercise). Exercise groups were trained by using a 6h/day swimming program. Muscles samples were harvested 18 hours after the last bout of exercise. The expression of pan-actin and β-tubulin were increased after prolonged endurance exercise (3 weeks) in skeletal muscle. The expression of GAPDH were decreased in acute exercise (1 day and 3 day). But, there was no change in the expression of β-actin in acute and prolonged exercise. These findings suggest that pan-actin and β-tubulin can only be used as a loading controls for the acute exercise (1-3 day) study, and GAPDH can only be use in prolonged exercise studies (more than 3 weeks). However, use of β-actin as a control in skeletal muscle is most preferable since the expression of this housekeeping protein is most stable regardless of duration of exercise training.
참고문헌 (18)
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