[关键词]
[摘要]
研究辣木(Moringa Oleifera. Lam)叶生物碱对宫颈癌Hela细胞增殖和凋亡的影响及可能机制。通过采用MTT法、克隆形成法、流式细胞术和western blot法检测经不同浓度辣木生物碱(20~320 μg/mL)处理48 h后的Hela细胞增殖、凋亡情况和蛋白表达水平。研究结果表明,随着辣木生物碱浓度的增加,Hela细胞的存活率逐渐下降,当辣木生物碱浓度为160 μg/mL时,细胞的存活率为35.87%(p<0.001),人正常结肠细胞NCW460的存活率为91.91%;克隆形成实验表明,辣木生物碱(40~160 μg/mL)显著抑制Hela细胞克隆形成,抑制率分别为26.04%(p<0.05)、37.19%(p<0.01)和67.77%(p<0.001);当浓度为80 μg/mL时,Hela细胞形态发生明显变化;进一步流式细胞术分析得知,Hela细胞内的凋亡细胞数随着辣木生物碱浓度的增加而增加,当细胞经160 μg/mL辣木生物碱处理48 h后,其细胞内凋亡细胞数为42.10%(p<0.001),通过对凋亡蛋白表达水平检测发现,Bax/Bcl-2比率和Caspase9表达水平随着辣木生物碱浓度的增加而增加,当辣木生物碱浓度达到80 μg/mL时,与对照相比有显著性差异。此外,辣木生物碱显著降低Stat3蛋白磷酸化水平和CyclinD1蛋白表达水平,当辣木生物碱浓度达到160 μg/mL时,与对照相比有显著性差异。以上实验结果表明,辣木生物碱具有抑制Hela细胞增殖和诱导细胞凋亡的作用,其机制可能与抑制Stat3信号通路活化相关。
[Key word]
[Abstract]
The aim of this study was to investigate the effects of Moringa Oleifera. Lam (M. oleifera) alkaloids on proliferation and apoptosis of cervical cancer Hela cells and its possible mechanism. Following 48h of treatment with M. oleifera alkaloids at various doses (20~320 μg/mL) , the proliferation, apoptosis and protein expression level of Hela cells were detected by MTT assay, colony formation assay, flow cytometric analysis and western blot. The results showed that the survival rate of Hela cells decreased gradually with the increase of M. oleifera alkaloids concentration. When the concentration of M. oleifera alkaloids was 160 μg/mL, the cell survival rate was 35.87% (p<0.001), the survival rate of human normal colon cell NCW460 is 91.91%. Colony formation experiments showed that M. oleifera alkaloids (40~160 μg/mL) significantly inhibited the colony formation of Hela cells, and the inhibition rates were 26.04% (p<0.05), 37.19% (p<0.01) and 67.77% (p<0.001), respectively. When the concentration of M. oleifera alkaloids was 80 μg/mL, the morphology of Hela cells changed significantly. Further flow cytometry analysis showed that the number of apoptotic cells in Hela cells increased with the increase of the concentration of M. oleifera alkaloid. When the cells were treated with 160 μg/mL of M. oleifera alkaloids for 48 h, the number of apoptotic cells was 42.1% (p<0.001). Further experiments showed that at 48 h, M. oleifera alkaloid up-regulated the ratio of Bax/Bcl-2 and the expressions of Caspase9 and down-regulated the expressions of P-Stat3 and CyclinD1 at the protein level in Hela cells. Our results indicate that M. oleifera alkaloids inhibits cell proliferation of Hela cells through the induction of apoptosis, which is mediated via inhibition of the Stat3-related pathway.
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[基金项目]
热作技术试验示范与服务支持—云南特色热带作物精深加工产品研发及示范项目(18190001);国家木薯产业技术体系项目(CARS-11-YNSJ);云南省中青年学术和技术带头人后备人才项目(2018HB040);云南省农业基础研究联合专项青年项目(2018FG001-088)