中国医师杂志
中國醫師雜誌
중국의사잡지
JOURNAL OF CHINESE PHYSICIAN
2013年
5期
581-585
,共5页
李科伟%黄薇%许志鹏%张晶晶%陈伟%张冬%黎志宏
李科偉%黃薇%許誌鵬%張晶晶%陳偉%張鼕%黎誌宏
리과위%황미%허지붕%장정정%진위%장동%려지굉
脂联素%骨肉瘤/病理学%蛋白激酶类/代谢%细胞增殖
脂聯素%骨肉瘤/病理學%蛋白激酶類/代謝%細胞增殖
지련소%골육류/병이학%단백격매류/대사%세포증식
Adiponectin%Osteosarcoma/pathology%Protein kinases/metabolism%Cell proliferation
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6 cm板上,接种密度l×105/ml,待细胞生长至约80%时,每板加入5 ml含脂联素的培养基(浓度1μg/ml),作用时间分别为0、15、30、60、120 min;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、10 μg/ml)脂联素重组体组,每组做5个平行孔,作用时间24 h,培养结束前4h,每孔加入10μl的CCK-8试剂,继续培养2.5h.490 nm测定光密度值(OD490).结果 脂联素浓度≥0.01 μg/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.
目的 研究脂聯素對MG-63細胞單燐痠腺苷激活的蛋白激酶(AMPK)燐痠化和細胞增殖的影響.方法 將骨肉瘤細胞株MG-63接種于6 cm闆上,接種密度l×105/ml,待細胞生長至約80%時,每闆加入5 ml含脂聯素的培養基(濃度1μg/ml),作用時間分彆為0、15、30、60、120 min;Western Blot檢測AMPK蛋白燐痠化水平,選擇最佳的脂聯素處理時間.分彆設對照組和脂聯素組(0、0.001、0.01、0.1、1μg/ml),根據脂聯素最佳處理時間處理後,Western Blot檢測AMPK燐痠化水平,確定脂聯素最佳作用濃度.依據最佳作用時間及最佳濃度,將PBS作為對照,相應濃度脂聯素作處理組,Western Blot檢測AMPK燐痠化水平,明確脂聯素對骨肉瘤MG-63細胞AMPK燐痠化的影響.CCK-8法檢測脂聯素對MG-63細胞增殖的影響,將MG-63細胞接種于96孔闆,每孔細胞數為5000箇,培養過夜.實驗分為空白對照組和5箇質量濃度梯度(0.001、0.01、0.1、1、10 μg/ml)脂聯素重組體組,每組做5箇平行孔,作用時間24 h,培養結束前4h,每孔加入10μl的CCK-8試劑,繼續培養2.5h.490 nm測定光密度值(OD490).結果 脂聯素濃度≥0.01 μg/ml時,MG-63細胞AMPK燐痠化水平顯著升高(t=5.894,P=0.007);短時間內脂聯素對MG-63細胞增殖無明顯抑製作用(F=6.335,P=0.072).結論 脂聯素可以增彊AMPK的燐痠化,短時間刺激脂聯素對MG-63細胞增殖無明顯抑製作用.
목적 연구지련소대MG-63세포단린산선감격활적단백격매(AMPK)린산화화세포증식적영향.방법 장골육류세포주MG-63접충우6 cm판상,접충밀도l×105/ml,대세포생장지약80%시,매판가입5 ml함지련소적배양기(농도1μg/ml),작용시간분별위0、15、30、60、120 min;Western Blot검측AMPK단백린산화수평,선택최가적지련소처리시간.분별설대조조화지련소조(0、0.001、0.01、0.1、1μg/ml),근거지련소최가처리시간처리후,Western Blot검측AMPK린산화수평,학정지련소최가작용농도.의거최가작용시간급최가농도,장PBS작위대조,상응농도지련소작처리조,Western Blot검측AMPK린산화수평,명학지련소대골육류MG-63세포AMPK린산화적영향.CCK-8법검측지련소대MG-63세포증식적영향,장MG-63세포접충우96공판,매공세포수위5000개,배양과야.실험분위공백대조조화5개질량농도제도(0.001、0.01、0.1、1、10 μg/ml)지련소중조체조,매조주5개평행공,작용시간24 h,배양결속전4h,매공가입10μl적CCK-8시제,계속배양2.5h.490 nm측정광밀도치(OD490).결과 지련소농도≥0.01 μg/ml시,MG-63세포AMPK린산화수평현저승고(t=5.894,P=0.007);단시간내지련소대MG-63세포증식무명현억제작용(F=6.335,P=0.072).결론 지련소가이증강AMPK적린산화,단시간자격지련소대MG-63세포증식무명현억제작용.
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 × 105 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,respectively ; 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 adiponectin.Based on the optimal processing time and optimal concentration,PBS was used as a control,the corresponding 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 phosphorylation of osteosarcoma MG-63 cells.CCK-8 assay was used to investigate the effect of adiponectin on the cell proliferation of MG-63 cells.The MG-63 cells were seeded in the 96-well plates (5,000 cells/well) and cultured 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 cells were cultured for another 2.5 h.The optical density (OD490) 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 significantly elevated (t =5.894,P =0.007).The short-time stimulation of adiponectin did not inhibit the proliferation of MG-63 cells (F =6.335,P =0.072).Conclusions Adiponectin can enhance the AMPK phosphorylation.The short-time stimulation of adiponectin did not inhibit the proliferation of MG-63 cells.