文章摘要
廖〓洋,陈建斌,卢前微,葛群芳,杨泽松.大蒜素诱导THP-1细胞凋亡的作用及机制[J].南京医科大学学报,2010,(2):155~160
大蒜素诱导THP-1细胞凋亡的作用及机制
Allicin induced apoptosis of THP-1cells and its mechanism
  
DOI:10.7655
中文关键词: 大蒜素  THP-1细胞  凋亡  细胞周期  Fas/FasL
英文关键词: allicin  THP-1 cells  apoptosis  cell cycle  Fas/FasL
基金项目:重庆市卫生局医学科学技术研究项目基金资助(07-2-035)
作者单位
廖〓洋 重庆医科大学附属第一医院血液内科,重庆〓400016 
陈建斌  
卢前微  
葛群芳  
杨泽松  
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中文摘要:
      目的:探讨大蒜素对急性单核细胞白血病细胞株THP-1凋亡及凋亡基因Fas/FasL表达的影响?方法:采用四甲基偶氮唑蓝(MTT) 法测定大蒜素对THP-1细胞增殖抑制率;AO/EB染色观察细胞凋亡形态;Annexin V-FITC/PI双染法流式细胞术(FCM)检测凋亡率的变化;FCM检测细胞凋亡率和细胞周期分布;免疫细胞化学法和Western blot观察细胞Fas?FasL蛋白表达?结果:MTT显示大蒜素能抑制THP-1细胞增殖,呈时间-剂量依赖性,72 h中效浓度(IC50)为12.8 mg/L?Annexin V-FITC/PI检测凋亡率随大蒜素浓度增加而增加?6.4?12.8 mg/L大蒜素作用THP-1细胞72 h后,FCM检测G0/G1期细胞比率明显上升,而S期比率明显下降,均出现凋亡峰,凋亡率分别为(22.93±2.93)%和(36.73±2.88)%,呈剂量依赖性,与对照组比较有显著差异(P < 0.01)?在荧光显微镜下可区分早?晚期凋亡细胞?活细胞和坏死细胞?免疫组化显示大蒜素作用后Fas/FasL蛋白上调,主要分布在细胞浆和细胞膜?Western blot结果显示Fas/FasL蛋白表达量随大蒜素浓度的升高而增加,呈剂量依赖性?0?6.4?12.8 mg/L大蒜素分别作用后Fas蛋白表达水平(相对灰度值)分别为0.287 4±0.009,0.427±0.008和0.597±0.011;FasL蛋白表达水平分别为0.212±0.014,0.299±0.028和0.409±0.027;差别均有显著性意义(P < 0.01)?结论:大蒜素可诱导THP-1细胞发生凋亡,诱导凋亡的机制可能部分与其上调Fas及FasL表达有关?
英文摘要:
      Objective:The purpose of this paper is to investigate the effects of allicin on the apoptosis of THP-1 cells and expressions of Fas/FasL. Methods:The proliferation inhibition rates of THP-1 cells after various treatments were examined by MTT assay. After the treatment of THP-1 cells by allicin,morphological change of apoptotic cells was investigated by AO/EB fluorescent staining under fluorescent microscope. Apoptosis rate was determined with Annexin V-FITC/PI double staining by flow cytometry. Flow cytometry was used to observe the effect of allicin on apoptosis and cell cycle distribution of THP-1 cells. The expression changes of Fas and FasL were detected by immunohistochemical staining. The changes of Fas/FasL proteins were detected by Western blot. Results:The proliferations of THP-1 cells were inhibited by allicin. MTT assay showed that the allicin inhibited the proliferation of the THP-1 cells,and the inhibition was dependent on both dose and time. The IC50 of 72 h was 12.8 mg/L. Apoptosis rate detected by Annexin V-FITC/PI was proportional to the concentration of the allicin. The percentage of G0/Gl phase cells was increased,but the S phase cell was decreased when treated with allicin for 72 h. 72 h after exposure to 6.4,12.8 mg/L allicin,the apoptotic rates of THP-1 cells were(22.93±2.93)% and(36.73±2.88)%,respectively. They were also dose dependent,and statistically significant when compared with the controls(P < 0.01). The cells could be classificated into:early apoptotic cells,late apoptotic cells,alive cells and dead cells under the fluorescent microscope. After the immunohistochemical staining test,the expression of Fas/FasL increased in THP-1cells was induced by allicin. Fas/FasL was located in the cytoplasm and cytolemma. Western blot test showed that the Fas/FasL protein expression was proportional to the concentration of allicin and it was also dose dependent. After treated with 0,6.4 and 12.8 mg/L allicin for 72 h,the levels of Fas proteins were 0.287±0.009,0.427±0.008 and 0.597±0.011,respectively and the levels of FasL proteins were 0.212±0.014,0.299±0.028 and 0.409±0.027,respectively. The difference was statistically significant(P < 0.01). Conclusion:Allicin induced apoptosis of human THP-1 cells is partly by up-regulating the expressions of Fas/FasL.
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