[1]于雅丽,王雷鸣.二氯乙酸钠对人骨肉瘤MG63细胞增殖、凋亡和迁移的影响[J].中医正骨,2017,29(01):11-17.
　YU Yali,WANG Leiming.Effect of sodium dichloroacetate on cell proliferation,apoptosis and migration in human MG63 osteosarcoma cells[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2017,29(01):11-17.
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二氯乙酸钠对人骨肉瘤MG63细胞增殖、凋亡和迁移的影响()
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《中医正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第29卷
期数:
2017年01期
页码:
11-17
栏目:
基础研究
出版日期:
2017-01-20

文章信息/Info

Title:
Effect of sodium dichloroacetate on cell proliferation,apoptosis and migration in human MG63 osteosarcoma cells
作者:
于雅丽; 王雷鸣
河南省郑州市骨科医院,河南 郑州 450052
Author(s):
YU Yali; WANG Leiming
Zhengzhou Orthopedic Hospital,Zhengzhou 450052,Henan,China
关键词:
骨肉瘤;  二氯乙酸盐;  细胞增殖;  细胞凋亡;  细胞运动;  半胱氨酸天冬氨酸蛋白酶3;  MG63细胞株
Keywords:
osteosarcoma;  dichloroacetate;  cell proliferation;  apoptosis;  cell movement;  caspase 3;  MG63 cell lines
摘要:
目的:观察二氯乙酸钠(sodium dichloroacetate,DCA-Na)对人骨肉瘤MG63细胞增殖、凋亡和迁移的影响。方法:将对数生长期的MG63细胞随机分为对照组和低、中、高浓度DCA-Na组,对照组不加DCA-Na,低、中、高DCA-Na组加入DCA-Na(终浓度分别为50 μg·mL-1、100 μg·mL-1、200 μg·mL-1),并设1个只加等量培养基不加细胞和DCA-Na的空白组。分别在干预24 h、48 h、72 h后,采用Cell Counting Kit-8细胞活性检测试剂盒分光光度法检测MG63细胞增殖情况,测定光密度(optical density,OD),计算低、中、高DCA-Na组细胞增殖抑制率,细胞增殖抑制率=[1-(DCA-Na组OD值-空白组OD值)/(对照组OD值-空白组OD值)]×100%; 分别采用Caspase-3活性检测试剂盒分光光度法和Annexin V-FITC细胞凋亡检测试剂盒流式细胞法检测MG63细胞Caspase-3酶活性情况和细胞凋亡情况; 并在干预48 h后采用Transwell实验检测MG63细胞迁移情况。结果:①细胞增殖检测结果。干预后,低、中、高DCA-Na组MG63细胞增殖抑制明显,且随干预时间的延长,3组细胞增殖抑制率均呈上升趋势。各时间点间MG63细胞增殖抑制率差异有统计学意义(F=847.080,P=0.000),存在时间效应; 干预24 h、48 h、72 h后,低、中、高DCA-Na组MG63细胞增殖抑制率的组间差异均有统计学意义,且低DCA-Na组<中DCA-Na组<高DCA-Na组[(10.802±3.000)%,(18.792±2.261)%,(27.080±3.133)%,F=2.795,P=0.000;(13.098±1.299)%,(25.215±2.676)%,(44.382±2.397)%,F=4.362,P=0.000;(14.728±1.177)%,(35.297±4.757)%,(64.227±4.549)%,F=5.896,P=0.000]; 3组间MG63细胞增殖抑制率总体比较,差异有统计学意义,存在分组效应(F=296.412,P=0.000); 时间因素与分组因素之间存在交互效应(F=75.678,P=0.000)。②Caspase-3酶活性检测结果。对照组MG63细胞Caspase-3酶活性无明显变化,干预后低、中、高DCA-Na组MG63细胞Caspase-3酶活性均呈上升趋势。各时间点间MG63细胞Caspase-3酶活性的差异有统计学意义(F=1 480.792,P=0.000),存在时间效应; 干预24 h、48 h、72 h后,4组间MG63细胞Caspase-3酶活性的差异均有统计学意义,且对照组<低DCA-Na组<中DCA-Na组<高DCA-Na组(0.027±0.003,0.143±0.005,0.153±0.008,0.161±0.003,F=2.320,P=0.000; 0.035±0.003,0.174±0.004,0.184±0.007,0.253±0.001,F=1.014,P=0.000; 0.031±0.004,0.246±0.006,0.275±0.003,0.371±0.004,F=1.000,P=0.000); 4组间MG63细胞Caspase-3酶活性总体比较,差异有统计学意义,存在分组效应(F=624.975,P=0.000); 时间因素与分组因素之间存在交互效应(F=94.579,P=0.000)。③流式细胞法细胞凋亡检测结果。对照组MG63细胞凋亡率无明显变化,干预后低、中、高浓度DCA-Na组MG63细胞凋亡率均呈上升趋势。各时间点间MG63细胞凋亡率的差异有统计学意义(F=359.645,P=0.000),存在时间效应; 干预24 h、48 h、72 h后,各组间MG63细胞凋亡率的差异均有统计学意义,且对照组<低DCA-Na组<中DCA-Na组<高DCA-Na组[(2.554±0.427)%,(10.708±2.012)%,(20.857±2.531)%,(27.312±2.140)%,F=6.733,P=0.000;(1.748±0.202)%,(18.604±2.721)%,(29.471±1.605)%,(36.873±2.734)%,F=9.292,P=0.000;(1.944±0.112)%,(24.071±3.921)%,(30.050±3.921)%,(38.211±1.721)%,F=8.237,P=0.000]; 4组间MG63细胞凋亡率总体比较,差异有统计学意义,存在分组效应(F=46.627,P=0.000); 时间因素与分组因素之间存在交互效应(F=7.012,P=0.000)。④细胞迁移检测结果。干预48 h后,4组迁移细胞数[显微镜每个视野下(×200)]的组间差异有统计学意义[(84.45±10.45)个,(74.56±9.45)个,(65.41±5.21)个,(40.21±4.52)个,F=148.243,P=0.000)]; 低、中、高DCA-Na组迁移细胞数均少于对照组(P=0.017,P=0.001,P=0.000),且低DCA-Na组>中DCA-Na组>高DCA-Na组(P=0.012,P=0.001,P=0.000)。结论:DCA-Na可抑制人骨肉瘤MG63细胞的增殖,增强MG63细胞Caspase-3酶活性,诱导和促进MG63细胞凋亡,抑制MG63细胞的迁移; 且浓度越高、干预时间越长,其影响越明显。
Abstract:
Objective:To explore the effect of sodium dichloroacetate(DCA-Na)on cell proliferation,apoptosis and migration in human MG63 osteosarcoma cells.Methods:The MG63 cells were randomly divided into control group,low-concentration DCA-Na group,middle-concentration DCA-Na group and high-concentration DCA-Na group in the logarithmic growth phase.No DCA-Na were placed in cells in control group,and DCA-Na with final concentration of 50,100 and 200 μg/mL were placed in cells in low-,middle- and high-concentration DCA-Na group respectively,meanwhile,a blank group was established with same amount of culture medium and without cells and DCA-Na.At 24,48 and 72 hours after the intervention,the MG63 cell proliferations were detected by using Cell Counting Kit-8 spectrophotometry and the optical density(OD)were measured for calculating the cell proliferation inhibition rate([1-(ODDCA-Na group-ODblank group)/(ODcontrol group-ODblank group)]×100%)of low-,middle- and high-concentration DCA-Na group respectively.Meanwhile,The Caspase-3 enzymatic activity and apoptosis of MG63 cells were detected by using Caspase-3 activity assay kit spectrophotometry and Annexin V-FITC apoptosis assays kit flow cytometry respectively.The MG63 cell migration was detected by using Transwell assay at 48 hours after the intervention.Results:The MG63 cell proliferations were obviously inhibited after intervention in low-,middle- and high-concentration DCA-Na group,and a time-dependent rising trend of cell proliferation inhibition rate was found in the 3 groups.There was statistical difference in the MG63 cell proliferation inhibition rate between different timepoints(F=847.080,P=0.000),in other words,there was time effect.There was statistical difference in the MG63 cell proliferation inhibition rate between low-,middle- and high-concentration DCA-Na group at 24,48 and 72 hours after the intervention,and the MG63 cell proliferation inhibition rate was lower in the low-concentration DCA-Na group compared to middle-concentration DCA-Na group and was lower in the middle-concentration DCA-Na group compared to high-concentration DCA-Na group(10.802+/-3.000,18.792+/-2.261,27.080+/-3.133%,F=2.795,P=0.000; 13.098+/-1.299,25.215+/-2.676,44.382+/-2.397%,F=4.362,P=0.000; 14.728+/-1.177,35.297+/-4.757,64.227+/-4.549%,F=5.896,P=0.000).There was statistical difference in MG63 cell proliferation inhibition rate between the 3 groups in general,in other words,there was group effect(F=296.412,P=0.000).There was interaction between time factor and group factor(F=75.678,P=0.000).No significant change was found in Caspase-3 enzymatic activity of MG63 cells in control group,while a rising trend of the Caspase-3 enzymatic activity of MG63 cells was found in low-,middle- and high-concentration DCA-Na group after intervention.There was statistical difference in the Caspase-3 enzymatic activity of MG63 cells between different timepoints(F=1 480.792,P=0.000),in other words,there was time effect.There was statistical difference in the Caspase-3 enzymatic activity of MG63 cells between the 4 groups at 24,48 and 72 hours after the intervention,and the Caspase-3 enzymatic activity of MG63 cells presented a low-to-high trend in control group and low-,middle- and high-concentration DCA-Na group in turn(0.027+/-0.003,0.143+/-0.005,0.153+/-0.008,0.161+/-0.003,F=2.320,P=0.000; 0.035+/-0.003,0.174+/-0.004,0.184+/-0.007,0.253+/-0.001,F=1.014,P=0.000; 0.031+/-0.004,0.246+/-0.006,0.275+/-0.003,0.371+/-0.004,F=1.000,P=0.000).There was statistical difference in Caspase-3 enzymatic activity of MG63 cells between the 4 groups in general,in other words,there was group effect(F=624.975,P=0.000).There was interaction between time factor and group factor(F=94.579,P=0.000).No significant change was found in MG63 cell apoptosis rate in control group,while a rising trend of MG63 cell apoptosis rate was found in low-,middle- and high-concentration DCA-Na group after intervention.There was statistical difference in the MG63 cell apoptosis rate between different timepoints(F=359.645,P=0.000),in other words,there was time effect.There was statistical difference in the MG63 cell apoptosis rate between the 4 groups at 24,48 and 72 hours after the intervention,and the MG63 cell apoptosis rate presented a low-to-high trend in control group and low-,middle- and high-concentration DCA-Na group in turn(2.554+/-0.427,10.708+/-2.012,20.857+/-2.531,27.312+/-2.140%,F=6.733,P=0.000; 1.748+/-0.202,18.604+/-2.721,29.471+/-1.605,36.873+/-2.734%,F=9.292,P=0.000; 1.944+/-0.112,24.071+/-3.921,30.050+/-3.921,38.211+/-1.721%,F=8.237,P=0.000).There was statistical difference in the MG63 cell apoptosis rate between the 4 groups in general,in other words,there was group effect(F=46.627,P=0.000).There was interaction between time factor and group factor(F=7.012,P=0.000).There was statistical difference in the number of migratory MG63 cells under the optical microscope(×200)between the 4 groups at 48 hours after the intervention(84.45+/-10.45,74.56+/-9.45,65.41+/-5.21,40.21+/-4.52,F=148.243,P=0.000).The migratory MG63 cells was less in low-,middle- and high-concentration DCA-Na group compared to control group(P=0.017,P=0.001,P=0.000),and the low-concentration DCA-Na group surpassed middle- and high-concentration DCA-Na group and middle-concentration DCA-Na group surpassed high-concentration DCA-Na group in the number of migratory MG63 cells(P=0.012,P=0.001,P=0.000).Conclusion:DCA-Na can inhibit the proliferation of human MG63 osteosarcoma cells,increase the Caspase-3 enzymatic activity of MG63 cells,induce and accelerate the apoptosis of MG63 cells and inhibit the migration of MG63 cells.Moreover,the higher the concentration of DCA-Na is and the longer the intervention time is,the more obvious the effect is.

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备注/Memo

备注/Memo:

通讯作者:王雷鸣 E-mial:wanglm3@126.com
更新日期/Last Update: 2017-01-20