代代花总黄酮对3T3-L1细胞增殖活性的影响  

Effects of the Total Flavonoids from Citrus aurantium on Proliferation and Apoptosis of 3T3-L1 Cells

作　　者：郝云芳[1] 杨丽[1] 姜建国[1] 

机构地区：[1]华南理工大学轻工与食品学院,广东广州510640

出　　处：《现代食品科技》2016年第9期35-40,266,共7页Modern Food Science and Technology

基　　金：国家自然科学基金青年项目(31301453)

摘　　要：本文研究了代代花总黄酮抑制3T3-L1细胞增殖及诱导其凋亡的作用。不同浓度的代代花总黄酮作用于3T3-L1细胞24 h之后,采用MTT法检测代代花总黄酮对细胞增殖的抑制作用;使用倒置显微镜观察细胞形态学的变化;Annexin V-EGFP/PI标记检测细胞凋亡率;PI标记法检测了代代花总黄酮对细胞周期的影响;活性氧(ROS)试剂盒检测细胞内ROS水平;荧光定量PCR检测了凋亡相关基因的m RNA水平表达。结果表明,高浓度(300μg/m L和400μg/m L)的代代花总黄酮可显著抑制3T3-L1细胞增殖,细胞的抑制率分别为38%和63%,且细胞形态发生了明显变化,并显著升高了细胞内ROS浓度。细胞凋亡实验结果显示,代代花总黄酮可诱导3T3-L1细胞早期凋亡和晚期凋亡,100μg/m L、200μg/m L和300μg/m L代代花总黄酮处理组的细胞早期凋亡率分别为4.6%、15.7%和22.5%;晚期凋亡率分别为14.4%、8.3%和32.2%。而细胞周期实验则表明,处理后3T3-L1细胞G0/G1期细胞数比例从58.9%下降到51.4%,而相应的S期细胞数比例呈小幅度增加,G2/M期细胞数比例变化不明显。凋亡相关基因p21及p53的m RNA表达明显升高及促凋亡基因bax与抗凋亡基因bcl-2比例的升高使3T3-L1进入细胞凋亡程序。The impact of the total flavonoids of Citrus aurantium（TFC） treatment on proliferation and apoptosis of 3T3-L1 cells was studied. After 3T3-L1 cells were cultured and treated with TFC in different concentrations for 24 h, the effect of TFC on cell proliferation was examined by 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide（MTT） assay. Morphological changes of 3T3-L1 cells were observed under an inverted microscope, the cell apoptotic rate was determined by Annexin V-EGFP/PI double staining, and propidium iodide（PI） staining was used to measure the effect of TFC on cell cycle. The intracellular reactive oxygen species（ROS） level was measured using a ROS assay kit, and the mRNA expression level of apoptosis-related genes was determined by fluorescence quantitative real-time polymerase chain reaction（RT-PCR）. The results showed that high concentrations of TFC（300 and 400 μg/mL） could significantly inhibit the proliferation of 3T3-L1 cells; the inhibition rates on cells were 38% and 63%, respectively, and the morphological characteristics of 3T3-L1 cells were changed. The ROS concentration in cells was also increased significantly. The apoptosis experiments indicated that TFC could induce early and late apoptosis of 3T3-L1 cells; the early apoptosis rates for the treatments with 100 μg/mL, 200 μg/mL and 300 μg/mL TFC for 24 h were 4.6%, 15.7% and 22.5%, respectively, and the late apoptosis rates were 14.4%, 8.3% and 32.2%, respectively. The cell cycle results showed that the G0/G1 phase ratio of the treated 3T3-L1 cells decreased from 58.9% to 51.4%%, the ratio of corresponding S phase was increased slightly, and the ratio of the cells in G2/M phase was not significantly changed. The increase in the ratio of pro-apoptotic gene bax and anti-apoptotic genebcl-2 and the increase in the mRNA expression of apoptosis-related genes p21 and p53 promoted apoptosis in 3T3-L1 cells.

关 键 词：代代花总黄酮 3T3-L1细胞 细胞凋亡 细胞周期 

分 类 号：R285[医药卫生—中药学]