蛴螬提取物对MCF-7人乳腺癌细胞株凋亡的影响

目的：研究蛴螬提取物对MCF-7人乳腺癌细胞株凋亡通路的影响，为天然药物蛴螬的开发提供实验和理论依据。方法:应用MTT法检测蛴螬提取物对MCF-7人乳腺癌细胞株的抗增殖作用及细胞毒活性；通过倒置显微镜、HE染色、吖碇橙（AO）/溴化乙啶（EB）荧光染色方法观察肿瘤细胞的形态学改变；应用流式细胞仪检测细胞凋亡率的变化；应用免疫细胞化学技术（S-P法）检测用药前后凋亡通路中Bcl-2、Fas、caspase-9、caspase-3的表达改变，探讨蛴螬提取物对凋亡通路的影响。结果:(1)用MTT法检测结果表明，蛴螬提取物在体外对MCF-7人乳腺癌细胞株有明显的抑制增殖作用，实验组与对照组相比其生长抑制率有显著差异（P<0.01），而且这种抑制作用呈现浓度和时间的依赖性；(2)倒置显微镜观察表明，实验组可见胞核固缩、碎裂、凋亡小体形成等凋亡形态学变化；(3)HE染色表明实验组胞核浓缩呈蓝黑色、胞浆呈淡红色、核染色质浓缩、呈碎块状，有凋亡小体形成；(4)经AO／EB荧光染色观察结果表明，实验组出现凋亡细胞；(5)流式细胞仪结果表明实验组凋亡率明显增加，呈时间依赖性；(6)免疫细胞化学染色结果表明，实验组 Bcl-2表达下调，Fas、caspase-3、caspase-9表达均上调，与对照组比差异显著（P<0.01）。结论:①蛴螬提取物在体外显著抑制MCF-7人乳腺癌细胞株增殖；②蛴螬提取物对MCF-7人乳腺癌细胞株凋亡通路的影响机制可能是通过下调Bcl-2，上调Fas、caspase-3 、caspase-9的蛋白表达而起作用，是经由细胞凋亡的线粒体途径和死亡受体途径来完成凋亡的启动和执行。

Effects of the grub extract on apoptosis of MCF-7 human breast cancer cell line

AIM: To investigate the apoptotic pathway of MCF-7 breast cancer induced by the grub extract in vitro.METHODS: MTT assay was used to determine the effect of the grub extract on proliferation of MCF-7 human breast cancer cell line and cell toxicity. Morphological changes of the apoptosis in cancer cells were observed by HE staining through invert microscope, light microscope, AO/EB double fluorescent staining under fluorescent microscope. FCM was used to assay the change of apoptotic rate. The expression of Bcl-2, Fas, caspase-9, caspase-3 in apoptotic pathway was detected with immunocytochemical method before and after exposure to the grub extract, and the effect of that on apoptotic pathway was explored.RESULTS: (1) The MTT test showed that the growth of MCF-7 human breast cancer cell line was significantly inhibited by the grub extract in dose and time dependent manners. The inhibitory rate in exposure group was significantly different from that in control group (P<0.01). (2) Morphological changes of apoptosis including nuclear condensation, fragment and apoptosis body formation were observed by invert microscope. (3) The MCF-7 human breast cancer cells in experimental group by HE staining showed nuclear condensation and blue-black, cytoplasm slight red, nuclear chromatin condensation and fragment shape, apoptosis body formations. (4) Apoptosis in the experimental group was observed by AO/EB double fluorescent staining under fluorescent microscope. (5) FCM assay indicated that apoptotic rate increased significantly in time dependent manner in experimental group. (6) The expression of Bcl-2 was down-regulated, while that of Fas, caspase-3, caspase-9 was up-regulated, compared with control group (P<0.01).CONCLUSION: (1) The proliferation of MCF-7 human breast cancer cell line can be inhibited significantly by the grub extract in vitro. (2)The mechanism of effect of the grub extract on MCF-7 human breast cancer cell line might be mediated by down-regulation of Bcl-2 and up-regulation of Fas, caspase-3, caspase-9. This type of apoptosis starting and performing is through death receptor pathway and mitochondrial pathway.