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目的探讨核转录因子NF-κB和Caspase-9在大豆异黄酮诱导人乳腺癌细胞MCF-7细胞凋亡的作用和可能的机制。方法大豆异黄酮处理MCF-7细胞后,采用流式细胞仪检测乳腺癌细胞凋亡、线粒体心磷脂的变化,及免疫荧光显微技术检测线粒体中细胞色素c释放情况;并应用酶联免疫吸附方法检测了细胞凋亡中Caspase-9及NF-κB活性变化。结果 80mg/L大豆异黄酮处理乳腺癌MCF-7细胞24h后,细胞发生凋亡,凋亡率为7.56%;随时间的延长凋亡率不断上升,与对照组比较差异有统计学意义(P<0.01);同时,细胞线粒体心磷脂丢失增多,即大豆异黄酮处理组中心磷脂含量降低的凋亡细胞所占比例为(21.33±5.38)%,高于对照组(4.34±1.85)%,差别有统计学意义(P<0.01)。大豆异黄酮处理后,线粒体细胞色素c释放增多,Caspase-9活性增加,24h其活性为(13.65±3.42)μmol(/L·h·mg)蛋白,并随时间的延长而增加。经大豆异黄酮处理后,乳腺癌细胞NF-κB活性被抑制,24hNF-κB活性为0.19±0.03,低于对照组0.21±0.02,差别有统计学意义(P<0.05)。结论大豆异黄酮触发了线粒体途径而诱导肿瘤细胞凋亡,其可能机制为引发线粒体心磷脂丢失,造成线粒内膜损伤,导致细胞色素c释放和Caspase-9激活,同时抑制NF-κB活性,最终诱导细胞发生凋亡。
Objective To investigate the effects and possible mechanisms of NF-κB and Caspase-9 on apoptosis induced by soybean isoflavone in human breast cancer cell line MCF-7. Methods After MCF-7 cells were treated with soy isoflavones, the apoptosis of breast cancer cells was detected by flow cytometry, the changes of mitochondrial cardiolipin and the release of cytochrome c in mitochondria were detected by immunofluorescence microscopy. The expression of cytochrome c was detected by enzyme-linked immunosorbent assay Methods The changes of Caspase-9 and NF-κB activity in apoptosis were detected. Results After treated with 80 mg / L soy isoflavone for 24 h, MCF-7 cells were treated with isoflavone for 7 days. The apoptosis rate was 7.56%. The apoptosis rate was increased with the prolongation of time. The difference was statistically significant (P <0.01). At the same time, the loss of mitochondrial cardiolipin increased (21.33 ± 5.38)% in the soybean isoflavone group (4.34 ± 1.85)%, which was significantly lower than that in the control group There was statistical significance (P <0.01). After soy isoflavone treatment, mitochondrial cytochrome c release increased, Caspase-9 activity increased 24h after its activity (13.65 ± 3.42) μmol (/ L · h · mg) protein, and increased with time. The activity of NF-κB in breast cancer cells was inhibited by soybean isoflavone, the activity of 24hNF-κB was 0.19 ± 0.03, which was lower than that of the control group (0.21 ± 0.02), the difference was statistically significant (P <0.05). Conclusion Soy isoflavones trigger the mitochondrial pathway to induce tumor cell apoptosis. The possible mechanism is that the loss of mitochondrial cardiolipin leads to the loss of mitochondrial inner membrane, resulting in the release of cytochrome c and the activation of Caspase-9, as well as the inhibition of NF-κB activity. Eventually inducing cell apoptosis.