刚地弓形虫培养上清对乳腺癌MCF-7细胞增殖及凋亡的影响

Effect of culture supernatant of Toxoplasma gondii on proliferation and apoptosis of human breast cancer cell line MCF-7

目的观察体外培养条件下弓形虫培养上清对乳腺癌细胞MCF-7细胞增殖及其诱导其凋亡的情况。方法取对数生长期的MCF-7细胞(浓度为5×104和5×105)分别接种于不同细胞培养板,加入相同体积不同浓度弓形虫速殖子培养上清(速殖子浓度分别为2×107/mL、4×107/mL、6×107/mL、8×107/mL)与培养液共同作用12h、24h、48h、72h,四甲基氮噻唑蓝(MTT)法检测吸光度(A490值)并计算抑制率;Annexin-v-FITC/PI染色细胞后上流式细胞仪检测细胞凋亡率;AO/EB染料染色作用后细胞在荧光显微镜下观察细胞形态改变情况并拍照;琼脂糖凝胶电泳观察细胞凋亡DNA条带。结果MTT法检测结果显示MCF-7细胞增殖抑制率随着上清浓度和作用时间增加均明显增大,各实验组抑制率与对照组比较以及对照组之间比较差异均有统计学意义(P<0.05)。流式细胞仪检测显示MCF-7细胞凋亡率随着上清浓度和作用时间增加明显增大,48h达到凋亡最高值(19.79%),48h后凋亡率开始下降,死亡率增加;荧光显微镜观察实验组培养48h的MCF-7细胞,出现细胞核固缩及凋亡小体,对照组未出现凋亡小体。琼脂糖凝胶电泳见DNA梯形条带。结论弓形虫速殖子对体外培养MCF-7细胞增殖有明显的抑制作用,并可诱导MCF-7细胞凋亡。

To investigate the inhibitive effect of culture supernatants of Toxoplasma f.ondii on proliferation and apoptosis of human breast cancer cell line MCF7, MCF7 cells （concentration of 5 × 10^4/mL, 5 × 10^5/mL） were harvested in different cell culture plates, and the cells were treated for different hours with different concentrations of Toxoplasrna culture supernarant （the concentration of tachyzoites was 2 × 10^7/mL, 4 × 10^7/ mL, 6 × 10^7/ mL, 8 × 10^7/ mL respectively）. Then the growth inhibition rate was measured with MTT method. The apoptosis was detected with fluorescence microscopy with AO/EB staining to observe the changes of MCF7cell morphology, agarose electrophoresis to detect the DNA changes of MCF7 cells, and FCM to observe the rates of apoptosis of the MCF7 cells stained with Annexin v-FITC/PI. It was found that the culture supernatant of Toxoplasma inhibited the proliferation of MCF-7 cells, and the inhibitive effects depended upon concentration and the treatment time of T. gondii. The cell contractions and apoptotie bodies in the treated MCF7 cells were observed under the fluorescence microscope, and DNA fragments in the treated cells were revealed by the agarose electrophoresis. In the flow cytometric analysis, the maximum apoptosis rate, 19.79 %, was detected at the right time of 48h ours treated with supernatant, but the apoptosis rates decreased and dead rate increased after 48hours. Thus,the culture supernatant of Toa：oplasma gondii reveals to be an inhibition effect on the proliferation and apoptosis of MCF-7 cells in vitro.