外源性S100A6对人骨肉瘤细胞株U2OS的增殖、凋亡及β-catenin表达的影响  

Effect of exogenous S100A6 on human osteosarcoma cell line U2OS proliferation,apoptosis and β-catenin expression

作　　者：陈英华[1] 卫佳[1] 李星星[1] 吴丽美[1] 马闻[1] 张彦[1] 何通川[2] 周兰[1] 

机构地区：[1]重庆医科大学生物医学工程系、重庆市生物医学工程学重点实验室、重庆市超声医学工程重点实验室-省部共建国家重点实验室培育基地,400016 [2]美国芝加哥大学医学中心分子肿瘤研究室

出　　处：《重庆医科大学学报》2009年第7期817-821,共5页Journal of Chongqing Medical University

基　　金：国家自然科学基金资助项目(30772548)

摘　　要：目的:探讨外源性S100A6对骨肉瘤细胞株U2OS的生物学作用及其可能机制。方法:MTT法检测S100A6对U2OS细胞增殖的影响及浓度依赖性,台盼蓝拒染法计数活细胞检测S100A6对U2OS增殖作用的时间依赖性,Hoechst染色检测细胞凋亡,Westernblot法及免疫细胞化学法检测细胞内β-catenin的表达变化。结果:S100A6作用3d时,浓度为30、100、300和1000μg/ml的重组S100A6(GST-S100A6)组的OD值分别比相应浓度的GST对照组的OD值减少20.5%、23.7%、32.2%和36.8%,P<0.05;蛋白浓度为100μg/ml时,GST-S100A6组的细胞数在前2d与GST组相似,第3、4、5d的细胞数分别降低到GST组的59%、46%、48%,P<0.05;GST-S100A6干预3d后细胞凋亡率增高3.16倍,P<0.01;免疫细胞化学法测得经GST-S100A6处理3d后β-catenin的平均光密度值比GST组增加22.9%,P<0.01;经携带S100A6基因的重组腺病毒(Ad-S100A6)处理3d后,Westernblot法检测细胞中β-catenin的校正灰度值为1.4204±0.1999,比对照组(Ad-GFP组,0.9996±0.1000)显著增高,P<0.01。结论:外源性S100A6能够抑制U2OS细胞的增殖、促进其凋亡和增加U2OS细胞中β-catenin水平。Objective： we investigated the biological effects of exogenous S100A6 on osteosarcoma cell line U2OS and explored its possible mechanism. Methods：MTT assay was used to determine S100A6 influence on U2OS cells proliferation and their conce ntration-dependent relationship. The trypan blue exclusion assay was used to observe S100A6 inhibitory effect on U2OS cells proliferation in a time-dependent manner; Hoechst staining was used to determine cell apoptosis;Western blot and immunocytochemical methods to determine expressions of eyto- β -catenin. Results：We found that , on the first three days, OD values in cells subjected to the recombinant protein S100A6（ GST-S100A6 ）with the concentrations of 30,100,300 and 1 000μ g/ml respectively were obviously lower than those in the GST group, by 20.5%, 23.7%, 32.2% and 36.8% accordingly （P 〈0.05 ）;when protein concentration was 100 μ g/ml, cell counting in GST-S100A6 group was similar with GST group in the first two days,while decreased significantly to 59% ,46% ,48% on the third, fourth,fifth day compared with the GST group （P 〈0.05）;cell apoptosis rates increased by 3.16 fold three days after subjected to GST-S100A6 （P 〈0.01 ）. Average optical density ofβ-catenin in GST- S100A6 group increased by 22.9% compared with the GST group （P 〈0.01 ） 72h after exposure, using immunocytochemical assay;72 h after treatment with the recombinant adenovirus carrying S100A6 gene （Ad-S100A6）, corrected gray value of intracellular β-catenin was 1.420 4 ±0.199 9, obviously higher than those in the control group 0.999 6 ± 0.100 0 （P〈0.01）.Conclusion： Exogenous protein S100A6 could inhibite U2OS cells proliferation,promote apoptosis and then increase intracellular β -catenin concentration.

关 键 词：S100A6 骨肉瘤 β—catenin 

分 类 号：R730.231[医药卫生—肿瘤]