亚健康疲劳状态时血清对骨骼肌细胞线粒体膜细胞色素C氧化酶活性和线粒体能量负荷的影响

Influence of sub-health fatigue serum on the activity of cytochrome C oxidase and energy charge of mitochondrial membrane of human skeletal muscle cells

背景:前期研究表明亚健康疲劳者血清对体外培养骨骼肌细胞的生存活力和细胞超微结构具有显著影响,实验进一步从能量代谢方面观察亚健康疲劳时血清对骨骼肌细胞的影响。目的:以亚健康状态时血清培养人骨骼肌细胞,观察线粒体膜细胞色素C氧化酶活性和能量负荷的变化,并与健康状态血清培养结果相比较。设计、时间及地点:对比观察实验,于2006-10/2007-01在南方医科大学中医证候研究实验室完成。材料:25～40岁符合中医相关男性亚健康疲劳状态诊断标准者10名,治疗前后采血分别制备亚健康疲劳状态血清和正常状态血清,人骨骼肌细胞购于美国Sciencell研究实验室。方法:将人骨骼肌细胞常规培养、细胞融合达80%以上时,按随机数字表法分为4组,20%亚健康状态血清组、30%亚健康状态血清组、20%正常状态血清组、30%正常状态血清组。分别用含不同浓度亚健康状态血清和正常状态血清DMEM/F12细胞培养液培养。主要观察指标:培养24,48,72h时,采用生物化学法和高效液相色谱法测定人骨骼肌细胞线粒体膜细胞色素C氧化酶活性和三磷酸腺苷、二磷酸腺苷、一磷酸腺苷含量。结果:含20%亚健康状态血清和含20%,30%正常状态血清DMEM/F12细胞培养液培养人骨骼肌细胞在72h内,细胞线粒体膜细胞色素C氧化酶活性和能量负荷各时点差异均无显著性意义(P>0.05),含30%亚健康状态血清DMEM/F12细胞培养液在培养人骨骼肌细胞48h和72h时,细胞线粒体膜细胞色素C氧化酶活性和能量负荷显著降低,与其他各时点比较,差异有显著性意义(P<0.05)。进一步分析显示,线粒体能量负荷与线粒体膜细胞色素C氧化酶活性呈正相关。结论:亚健康疲劳状态时血清可致人骨骼肌细胞呈现能量代谢障碍的表现。

BACKGROUND： Our previous study showed sub-health fatigue serum can notably impair the viability of human skeletal muscle ceils （HSKMCs） and ultrastructure of HSKMCs in vitro. We have observed the effect of sub-health fatigue serum on HSKMCs from the view of energy metabolism. OBJECTIVE： To study the influence of sub-health fatigue serum on the activity of cytochrome C oxidase （COX） and energy charge （EC） in HSKMCs mitochondria and to compare to the results of healthy serum. DESIGN, TIME AND SETTING： The randomized controlled experiment was performed at the Lab of Traditional Chinese Medicine Syndrome Research of Southern Medical University from October 2006 to January 2007. MATERIALS： The vein blood was collected to harvest serum from 10 male cases with sub-health, who are between 25 and 40 year old. Human HSKMCs were gained from Sciencell Research Laboratory of America. METHODS： When 80% confluence, HSKMCs were randomly divided into 20% sub-health serum group, 30% sub-health serum group, 20% normal serum group and 30% normal serum group. HSKMCs were cultured respectively with respective cell culture medium in vitro. The cells were incubated in DMEM/F12 supplemented with different concentrations of sub-health serum and normal MAIN OUTCOME MEASURES： Activity of COX, contents of adenosine triphosphate, adenosine diphosphate and adenosine monophosphate in HSKMC mitochondria were measured by biochemistry and high performance liquid chromatography （HPLC） at 24 hours, 48 hours and 72 hours after culturing cells. RESULTS： No significant difference in activity of COX and EC was detected after 72 hours of culturing HSKMCs in DMEM/F12 supplemented with 20% sub-health serum, 20%, 30% normal serum （P 〉 0.05）. After 48 and 72 hours of culturing HSKMCs in DMEM/F12 supplemented with 30% sub-health serum group, the activity of COX and EC were lowered significantly, there was significant difference in comparison with other time points （P 〈 0.05）. The value of EC was positively related to the activity of COX. CONCLUSION： Sub-health fatigue serum can induce energy metabolic block of HSKMCs.