大鼠H9c2心肌细胞有氧氧化及无氧酵解的影响因素探讨
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摘要
目的利用细胞能量代谢检测系统(seahorse XF analyzer)探讨H9C2大鼠心肌细胞能量代谢的影响因素。方法 H9c2大鼠心肌细胞贴壁培养后以不同细胞密度接种于Seahorse XF24孔板,利用糖酵解压力试剂盒检测糖酵解压力曲线;以105/mL密度接种细胞,以不同浓度碳酰氰-4-三氟甲氧基苯胺(FCCP)刺激细胞,利用线粒体有氧氧化试剂盒检测线粒体功能。结果各密度下细胞反应良好,以2×104细胞数对寡霉素刺激后的速率数值较好,为50～400范围,是较合适的细胞密度;2μmol/L FCCP能刺激最大反应,为合适的FCCP浓度。结论细胞密度和FCCP浓度是影响心肌细胞有氧氧化及无氧酵解功能测定的主要因素,H9c2心肌细胞以2×104/孔细胞密度接种,FCCP浓度2μmol/L刺激细胞将适合细胞线粒体功能的检测。
Objective This study was designed to explore the influence factors on H9 c2 cells energy metabolism with cellular energy metab-olism detection system(Seahorse XF Analyzer).Methods Different cell density of H9 c2 rat myocardial cell were cultured in SeahorseXF24 orifice plate,glycolysis was detected by glycolysis kits.With 105/mL cell density,different concentration FCCP stimulate cells,mito-chondrial function was detected using mitochondrial aerobic oxidation kits.Results The density of cells respond well to 2×104 cell numberof oligomycin stimulation rate value was better.50-400 range was the more appropriate cell density.2μmol/L FCCP could stimulate themaximum reaction,which was more appropriate concentration.Conclusion The cell density and FCCP concentration are the main factorsinfluencing the determination of cardiomyocytes glycolysis and mitochondrial respiratory.H9 c2 cardiomyocytes in 2×104/well cell inoculationdensity,and FCCP concentration 2μmol/L stimulate cells will be suitable for mitochondrial function test.
Objective This study was designed to explore the influence factors on H9 c2 cells energy metabolism with cellular energy metab-olism detection system(Seahorse XF Analyzer).Methods Different cell density of H9 c2 rat myocardial cell were cultured in SeahorseXF24 orifice plate,glycolysis was detected by glycolysis kits.With 105/mL cell density,different concentration FCCP stimulate cells,mito-chondrial function was detected using mitochondrial aerobic oxidation kits.Results The density of cells respond well to 2×104 cell numberof oligomycin stimulation rate value was better.50-400 range was the more appropriate cell density.2μmol/L FCCP could stimulate themaximum reaction,which was more appropriate concentration.Conclusion The cell density and FCCP concentration are the main factorsinfluencing the determination of cardiomyocytes glycolysis and mitochondrial respiratory.H9 c2 cardiomyocytes in 2×104/well cell inoculationdensity,and FCCP concentration 2μmol/L stimulate cells will be suitable for mitochondrial function test.
引文
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[11]王显,朱明军,孙建芝,等.“心衰康”对电镜下CHF模型心肌线粒体的影响[J].上海中医药大学学报,2000,14(1):33-35.
[12]晋献春,孙建芝,王显,等.心衰康对实验性兔心力衰竭心肌细胞膜Na+、K+-ATP酶的影响[J].中国中医药科技,1996,3(6):26.
[13]晋献春,孙建芝,王显,等.心衰康冲剂对实验性兔心力衰竭心功能及心钠素的影响[J].中国中西医结合杂志,1996,16(7):424-426.
[14]EIRIN A,EBRAHIMI B,KWON S H,et al.Restoration of mitochondrial cardiolipin attenuates cardiac damage in swine renovascular hypertension[J].J Am Heart Assoc,2016,5(6):e003118.
[15]GUO S,OLM-SHIPMAN A,WALTERS A,et al.A cell-based phenotypica ssayto identify cardioprotective agents[J].Circ Res,2012,110(7):948-957.
[2]DOENST T,NGUYEN T D,ABEL E D.Cardiac metabolism in heart failure:implications beyond ATP production[J].Circ Res,2013,113(6):709-724.
[3]陈灏珠.实用心脏病学[M].4版.上海:上海科学技术出版社,2007:688-737.
[4]GOFFART S,VON KLEIST-RETZOW J C,WIESNER R J.Regulation of mitochondrial proliferation in the heart:power-plant failure contributes to cardiac failure in hypertrophy[J].Cardiovasc Res,2004,64(2):198-207.
[5]VAN BILSEN M,SMEETS P J,GILDE A J,et al.Metabolic remodeling of the failing heart:the cardiac burn-out syndrome?[J].Cardiovasc Res,2004,61(2):218-226.
[6]TUUNANEN H,KNUUTI J.Metabolic remodeling in human heart failure[J].Cardiovasc Res,2011,90(2):251-257.
[7]吴华芹,胡元会,王阶,等.心复康口服液对心梗后心衰大鼠心肌组织AT含量及GLUT1基因表达的影响[J].中国中医基础医学杂志,2008,14(4):268-270.
[8]戎靖枫,周华,郭蔚,等.益气温阳方对心力衰竭大鼠心脏血流动力学及心肌能量代谢的影响[J].上海中医药大学学报,2012,26(5):81-84.
[9]王永霞,任红杰,朱明军,等.参附益心颗粒对慢性心力衰竭大鼠血清心钠素、脑钠素的影响[J].中国实验方剂学,2011,17(4):118-120.
[10]王永霞,张群生,任红杰,等.参附益心颗粒对慢性心力衰竭大鼠循环与心肌血管紧张素Ⅱ的影响[J].中国实验方剂学杂志,2012,18(21):226-229.
[11]王显,朱明军,孙建芝,等.“心衰康”对电镜下CHF模型心肌线粒体的影响[J].上海中医药大学学报,2000,14(1):33-35.
[12]晋献春,孙建芝,王显,等.心衰康对实验性兔心力衰竭心肌细胞膜Na+、K+-ATP酶的影响[J].中国中医药科技,1996,3(6):26.
[13]晋献春,孙建芝,王显,等.心衰康冲剂对实验性兔心力衰竭心功能及心钠素的影响[J].中国中西医结合杂志,1996,16(7):424-426.
[14]EIRIN A,EBRAHIMI B,KWON S H,et al.Restoration of mitochondrial cardiolipin attenuates cardiac damage in swine renovascular hypertension[J].J Am Heart Assoc,2016,5(6):e003118.
[15]GUO S,OLM-SHIPMAN A,WALTERS A,et al.A cell-based phenotypica ssayto identify cardioprotective agents[J].Circ Res,2012,110(7):948-957.