脂联素对骨肉瘤MG-63细胞增殖和AMPK磷酸化的影响  

Effects of adiponectin on the phosphorylation of adenosine monophosphate-activated protein kinaseand the cell proliferation of osteosarcoma MG-63 cells

作　　者：李科伟[1] 黄薇[2] 许志鹏[2] 张晶晶[2] 陈伟[3] 张冬 黎志宏[3] 

机构地区：[1]郑州市骨科医院,郑州450000 [2]中南大学代谢内分泌研究中心 [3]中南大学湘雅二医院骨科 [4]湖南省长沙市芙蓉区医院外科

出　　处：《中国医师杂志》2013年第5期581-585,共5页Journal of Chinese Physician

基　　金：国家自然科学基金项目（30800572）

摘　　要：目的研究脂联素对MG-63细胞单磷酸腺苷激活的蛋白激酶（AMPK）磷酸化和细胞增殖的影响。方法将骨肉瘤细胞株MG-63接种于6cm板上，接种密度1×10^5／ml，待细胞生长至约80％时，每板加入5ml含脂联素的培养基（浓度1μg／ml），作用时间分别为0、15、30、60、120min；Western Blot检测AMPK蛋白磷酸化水平，选择最佳的脂联素处理时间。分别设对照组和脂联素组（0、0．001、0．01、0．1、1μg／ml），根据脂联素最佳处理时间处理后，Western Blot检测AMPK磷酸化水平，确定脂联素最佳作用浓度。依据最佳作用时间及最佳浓度，将PBS作为对照，相应浓度脂联素作处理组，Western Blot检测AMPK磷酸化水平，明确脂联素对骨肉瘤MG-63细胞AMPK磷酸化的影响。CCK-8法检测脂联素对MG-63细胞增殖的影响，将MG-63细胞接种于96孔板，每孔细胞数为5000个，培养过夜。实验分为空白对照组和5个质量浓度梯度（0．001、0．01、0．1、1、10μg／ml）脂联素重组体组，每组做5个平行孔，作用时间24h，培养结束前4h，每孔加入10μl的CCK-8试剂，继续培养2．5h。490nm测定光密度值（OIM90）。结果脂联素浓度≥0．01μg／ml时，MG-63细胞AMPK磷酸化水平显著升高（t=5．894，P=0．007）；短时间内脂联素对MG-63细胞增殖无明显抑制作用（F=6．335，P=0．072）。结论脂联素可以增强AMPK的磷酸化，短时间刺激脂联素对MG-63细胞增殖无明显抑制作用。Objective To investigate the effect of adiponectin on the phosphorylation of adenosine monophosphate-activated protein kinase （AMPK） and the cell proliferation of osteosarcoma MG-63 cells. Methods The MG-63 cells were seeded in 6-cm board with a inoculum density of 1×10^5 cells/ml. When the cells grew up to about 80%, a volume （5 ml） of medium containing adiponectin （concentration 1 μg/ ml） was added to each plate and incubated for 0 min, 15 min, 30 min, 60 min, and 120 min, respective- ly; and Western Blotting was used to detect the levels of AMPK phosphorylation, and the optimal time point processed by adiponectin was selected. The control and adiponectin groups were set （0, 0. 001, 0.01, 0. 1, 1 μg/ml） according to the optimal processing time of adiponectin, respectively; and Western blotting was used to detect the levels of AMPK phosphorylation and determine the optimal concentration of adiponec- tin. Based on the optimal processing time and optimal concentration, PBS was used as a control, the corre- sponding concentrations of adiponectin were used as the experimental group. Western blotting was used to detect the levels of AMPK phosphorylation to determine the effect of adiponectin on AMPK phosphorylationof osteosarcoma MG-63 cells. CCK-8 assay was used to investigate the effect of adiponectin on the cell pro- liferation of MG-63 cells. The MG-63 cells were seeded in the 96-well plates （5,000 cells/well） and cul- tured overnight. The experiment was set as blank control group and adiponectin-recombinant groups with 5 different concentration gradients （0. 001, 0.01,0.1,1,10 μg/ml）. Five parallel wells were set for each group, and the cells were cultured for 24 h. During the last 4 h of culturing, a volume （ 10 μl） of CCK-8 reagent was added to each well, and the ceils were cultured for another 2. 5 h. The optical density （0D490） was obtained. Results When the concentration of adiponectin was greater than 0.01 μg/ml, the level of AMPK phosphorylation in MG-63 cells were si

关 键 词：脂联素 骨肉瘤 病理学 蛋白激酶类 代谢 细胞增殖 

分 类 号：R738.1[医药卫生—肿瘤]