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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。
This paper studied the generation of flower flavonoids 3T3-L1 cell proliferation and induction of apoptosis. After treated with different concentrations of total flavonoids of generational flowers for 24 h, MTT assay was used to detect the inhibitory effect of total flavonoids of generational flowers on cell proliferation. The morphological changes of cells were observed by inverted microscope. Annexin V-EGFP / PI The apoptosis rate of the apoptosis-related genes was detected by PI assay, ROS level was detected by ROS assay, and the m RNA level was detected by fluorescence quantitative PCR . The results showed that the total flavonoids of generational flowers with high concentration (300μg / ml and 400μg / ml) could significantly inhibit the proliferation of 3T3-L1 cells with the inhibitory rates of 38% and 63%, respectively, and the cell morphology changed obviously , And significantly increased intracellular ROS concentration. The results of apoptosis test showed that the total flavonoids of generation flowers could induce the early apoptosis and the late apoptosis of 3T3-L1 cells. In the early stage of the cells treated with 100μg / ml, 200μg / ml and 300μg / ml L, The apoptotic rates were 4.6%, 15.7% and 22.5% respectively, and the late apoptosis rates were 14.4%, 8.3% and 32.2% respectively. However, the cell cycle assay showed that the proportion of cells in G0 / G1 phase decreased from 58.9% to 51.4% in 3T3-L1 cells after treatment, while the corresponding proportion of S phase cells increased slightly while the proportion of G2 / M phase cells did not change obvious. Apoptosis-related genes p21 and p53 m RNA expression was significantly increased and the pro-apoptotic gene bax and anti-apoptotic gene bcl-2 increased the ratio of 3T3-L1 into the apoptosis program.