摘要
目的研究血清剥夺对心肌细胞增殖和凋亡的影响及其机制。方法将人心肌细胞AC16分为血清剥夺组(细胞在无胎牛血清的DMEM培养液中培养48 h)、对照组(细胞在含10%胎牛血清的DMEM培养液中培养48 h)。采用CCK-8法检测心肌细胞活力,EdU实验法检测细胞DNA合成水平,流式细胞术检测细胞凋亡水平及线粒体膜电位变化,蛋白质印迹分析检测心肌细胞中caspase-9、Bcl-2、P53、增殖细胞核抗原(PCNA)、cyclinD1、β-连环蛋白(β-catenin)、Wnt5a、Axis抑制蛋白1(Axin1)、蓬乱蛋白2(Dvl2)、蓬乱蛋白3(Dvl3)、低密度脂蛋白受体相关蛋白6(LRP6)的表达水平。结果 CCK-8实验结果显示对照组的光密度值为1.93±0.01,血清剥夺组的光密度值为1.08±0.08,后者为前者的55.8%,两组比较差异有统计学意义(P<0.01);细胞DNA合成受到抑制,血清剥夺组细胞DNA合成水平下降为对照组的65.6%,两组比较差异有统计学意义(P<0.05)。血清剥夺诱导了心肌细胞的凋亡,对照组凋亡率为(6.34±0.47)%,血清剥夺组凋亡率为(56.83±1.90)%,后者为前者的8.9倍,两组比较差异有统计学意义(P<0.01);血清剥夺组心肌细胞线粒体膜电位下降,但与对照组比较差异无统计学意义(P>0.05)。蛋白质印迹分析结果显示,血清剥夺促进了心肌细胞中caspase-9蛋白的表达,同时抑制了心肌细胞中Bcl-2、P53、PCNA、cyclin D1蛋白的表达;血清剥夺抑制了心肌细胞中β-catenin、Wnt5a、Axin1、Dvl2、Dvl3、LRP6蛋白的表达。结论血清剥夺可抑制心肌细胞增殖并促进细胞凋亡,其机制可能与抑制Wnt/β-catenin信号通路有关。
Objective To explore the effect of serum deprivation on proliferation and apoptosis of cardiomyocytes and its mechanisms.Methods Human cardiomyocytes AC16 were randomly divided into serum-deprivation group(the cells were cultured in DMEM medium without fetal bovine serum for 48 h)and control group(the cells were cultured in DMEM medium containing 10%fetal bovine serum for 48 h).CCK-8 assay was performed to test the proliferation ability,and EdU imaging was conducted to detect the DNA synthesis level.Flow cytometry was performed to analyze the apoptosis level and mitochondrial membrane potential.Western blotting was conducted to detect the protein expression levels of caspase-9,Bcl-2,P53,proliferating cell nuclear antigen(PCNA),cyclin D1,β-catenin,Wnt5a,Axis inhibition protein 1(Axin1),dishevelled 2(Dvl2),dishevelled 3(Dvl3)and low-density lipoprotein receptor-related protein 6(LRP6).Results CCK-8 assay showed that the optical density value of the control group was 1.93±0.01 and that of the serum-deprivation group was 1.08±0.08,which was 55.8%of the control group,and there was significant difference between the two groups(P<0.01).DNA synthesis was inhibited by serum deprivation,and the DNA synthesis level of the serum-deprivation group was 65.6%of the control group,with significant difference found between the two groups(P<0.05).Serum deprivation could induce cardiomyocyte apoptosis;the apoptotic rate of control group was(6.34±0.47)%,and that of serum-deprivation group was(56.83±1.90)%,which was 8.9 times of the control group,and there was significant difference between the two groups(P<0.05).Serum deprivation could decrease the mitochondrial membrane potential of cardiomyocytes,while there was no significant difference between the serum-deprivation and control groups(P>0.05).Western blotting analysis showed that serum deprivation could upregulate the protein expression of caspase-9 and downregulate the protein expression of Bcl-2,P53,PCNA and cyclin D1.Serum deprivation could inhibit the protein expression o
作者
周思维
王宇
左权
赵峰
王鹏贺
张莎
朱霓
赵仙先
吴弘
ZHOU Si-wei;WANG Yu;ZUO Quan;ZHAO Feng;WANG Peng-he;ZHANG Sha;ZHU Ni;ZHAO Xian-xian;WU Hong(Department of Cardiovasology,Changhai Hospital,Naval Medical University (Second Military Medical University),Shanghai 200433,China;Department of Cardiovasology,Shanghai Zhongye Hospital,Shanghai 200941,China)
出处
《第二军医大学学报》
CAS
CSCD
北大核心
2019年第2期157-161,共5页
Academic Journal of Second Military Medical University
基金
上海市卫生和计划生育委员会科研项目(201540217)~~
