【作者】 金华； 孙抒； 于柏艳； 杨万山；

【Author】 Jin Hua Sun Shu YuBai-Yan Yang Wan-Shan Department of Pathology, Yanbian University, Medical School, Yanji 133000, Jilin Province, P. R. of China.

【机构】 延边大学基础医学院病理学教研室； 延边大学肿瘤研究中心；

【摘要】 目的:研究蛴螬提取物对MCF-7人乳腺癌细胞株凋亡通路及细胞周期的影响,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,应用流式细胞仪检测细胞凋亡和细胞周期的变化,并应用免疫细胞化学技术(S-P 法)检测用药前后PCNA、Ki-67、CyclinD1的表达改变。结果: (1)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性差异(P<0．001),(2)倒置显微镜观察结果表明, 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染色结果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、可呈碎块状,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)免疫组化结果表明,实验组Bcl-2、Ki-67、 PCNA、CyclinD1表达均下调,Fas、Caspase-3、Caspase- 9表达均上调,与对照组有显著性差异(p<0．001),(6)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, 且随着蛴螬提取物作用时间的延长,S期细胞比率增多,实验组与对照组S期细胞比率相比明显增加(p<0．001),表明肿瘤细胞在S期聚积,使细胞在S期聚积,阻滞了向G2／ G0期的转换,从而影响细胞周期。结论:1．蛴螬提取物在体外对MCF-7人乳腺癌细胞株有显著的抑制增殖作用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。3．蛴螬提取物对 M C F-7人乳腺癌细胞株细胞周期的影响机制可能与调 CyclinD1、Ki-67、PCNA的蛋白表达有关,并且可以使该肿瘤细胞在S期聚集,阻止了肿瘤细胞向G2／G0期的转换, 从而干扰细胞周期的进程。更多还原

【Abstract】 Objective: To investigate the apoptotic pathway and the effect of cell cycle of MCF-7 breast cancer induced by the grub extract in vitro and provide with evidence of experiment and theory for the development of natural medicine. Methods: MTT assay was used to determine the effect of the grub extract anti-proliferation on MCF-7 human breast cancer cell line and cell toxicity. Morphological changes of the apoptosis of cancer cells were observed by HE staining through invert microscope 、light microscope, AO/EB double fluorescent staining under fluorescent microscope. The expression of Bcl-2, Fas, Caspase-9, Caspase-3 in apoptotic pathway was detected with im-munocytochemistry before and after exposure to the grub extract, and the effect of that on apoptotic pathway was explored. FCM was used to assay cell cycle of MCF-7 cells. Furthermore, the expression of PCNA, Ki-67 and CyclinD1 was detected with immunocytochemistry before and after exposure to the grub extract, and the possible mechanism of that on cell cycle was investigated. Results: (1) The MTT test showed that MCF-7 human breast cancer cell line growth was significantly inhibited by the grub extract in dose and time dependent manner. The inhibitory rate of exposure proup was significantly different from that of control group (P<0.001), (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, their apoptosis body formations, (4) Apoptosis in the experimental group was observed by AO/EB double fluorescent staining under fluorescent microscope, (5) The expression of Bcl-2, Ki-67, PCNA, cyclinDl was down-regulated, while that of Fas, Caspase-3, Caspase-9 was up-regulated, compared with control group( P<0.001 ). (6) FCM assay indicated that apoptotic rate increased significantly in time dependent manner in experimental group. Along with the action time extending of the grub extract,it indicated that the cells were cumulated in S phase compared with control group( P<0.001 ),and blocked the cell transmission to G2/ GO, then it influenced the cell cycle finally. Conclusion: 1. The proliferation of MCF-7 human breast cancer cell line was 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. 3. The mechanism of effect of the grub extract on MCF-7 human breast cancer cell line might be related with down-regulation of CyclinDl, Ki-67, PCNA and the grub extract induced cancer cells accumulation in S phase and blocked cells transmissi更多还原