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mRNA水平表达。结果表明，高浓度（300 μg/mL和400 μg/mL)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，100 μg/mL、200 μg/mL和300 μg/mL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mRNA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 gene bcl-2 and the increase in the mRNA expression of apoptosis-related genes p21 and p53 promoted apoptosis in 3T3-L1 cells.

国家自然科学基金青年项目（31301453）