粉防已碱抗乳腺肿瘤作用及其机制研究

【作者】 李桃花；

【导师】 李曰庆； 裴晓华；

【作者基本信息】 北京中医药大学 ， 中医外科学， 2010， 博士

【摘要】 1目的乳腺癌是成年女性最常见的恶性肿瘤之一。大约1／3乳腺癌患者呈雌激素受体(ER)阴性,与ER阳性乳腺癌比较,其恶性程度较高,对化疗及内分泌治疗不敏感,预后更差,目前没有理想的治疗方法。ER阴性乳腺癌的治疗是目前临床的难点,因此本研究探讨粉防已碱对ER阴性人乳腺癌MDA-MB-435S细胞作用及其机制,以及对人乳腺癌细胞MCF-7荷瘤裸鼠作用及其机制,以为临床治疗奠定进一步研究的基础。2方法2.1体外实验部分用MTT方法检测粉防已碱7个不同浓度组干预乳腺癌MDA-MB-435S细胞24h、48h、72h、96h后细胞的光吸收值(OD),划细胞生长曲线,并计算各组细胞抑制率及半数抑制浓度(IC50),说明粉防已碱对人乳腺癌MDA-MB-435S细胞体外生长的抑制作用。碘化丙啶(PI)单染流式细胞术测定法进行粉防已碱4个不同浓度组对人乳腺癌MDA-MB-435S细胞周期分析,观察不同浓度粉防已碱干预24h后各组G0／G1期、G2M期、S期的细胞百分比分布。利用Annexin V-FITC、PI双标记细胞凋亡检测法检测粉防已碱4个不同浓度组干预人乳腺癌MDA-MB-435S细胞24h、48h后对细胞凋亡的影响。2.2体内实验部分建立裸鼠人乳腺癌MCF-7细胞移植瘤动物模型,把24只成瘤动物随机分为模型组、粉防已碱组、阳性对照组,每组8只,共给药15天。治疗期间观察并记录各组裸鼠移植瘤的生长情况及移植瘤体积,药物处理结束后1天,处死裸鼠,称各组裸鼠移植瘤重,并计算抑瘤率。利用光镜及透射电镜观察各组移植瘤组织病理学变化和超微结构变化。利用图像定量分析方法观察各组移植瘤组织的Bcl-2、Bax、Survivin及VEGF蛋白表达水平。3结果3.1体外实验部分粉防已碱7个不同浓度组(Tet-0.5组、Tet-1组、Tet-2组、Tet-4组、Tet-6组、Tet-8组、Tet-16组)与同时间点正常对照组(control-1)比较OD值显著降低(P<0.05或0.01),并且随着粉防已碱浓度增加,其OD值降低。同一时间点的药物的抑制率随着粉防已碱浓度的增加而增大,存在量效关系。随着时间的延长,粉防已碱对MDA-MB-435S细胞体外生长的抑制作用增大,存在时间效应。粉防已碱作用于MDA-MB-435S细胞,剂量与时间之间存在交互作用(P<0.01)。24h、48h、72h、96h的IC50值分别为8.748μg/ml,3.585μg/ml、1.451μg/ml、1.117μg/ml。粉防已碱4个不同浓度组(Tet-0.5组、Tet-1组、Tet-2组、Tet-4组)作用于MDA-MB-435S乳腺癌细胞24h后,与同期正常对照组比较,s期、G2M期细胞比例均明显降低,差异具有统计学意义(均P<0.05),并且随粉防已碱浓度增加,S期、G2M期细胞比例降低；G0／G1期细胞比例均显著增高,差异具有统计学意义(均P<0.05),并且随粉防已碱浓度增加,G0／G1期细胞比例增高。作用24h后粉防已碱4个不同浓度组(Tet-0.5组、Tet-1组、Tet-2组、Tet-4组)的早期凋亡率与control-1组比较均增多,差异具有统计学意义(均P<0.05)；48h后与control-1组比较,Tet-1组、Tet-2组、Tet-4组的早期凋亡率显著增多(均P<0.05)；24h、48h早期凋亡率与药物浓度呈正相关(均P<0.05)；并且随作用时间延长,诱导早期凋亡率增加。24h后粉防已碱组晚期凋亡率与control-1组比较无差异(均P>0.05)；随作用时间延长,48h后粉防已碱高浓度组的晚期凋亡率逐渐增多,与control-1组比较差异有统计学意义(均P<0.05)。3.2体内实验部分用药第6天以后粉防已碱组和阳性对照组移植瘤体积与同一天的模型组比较显著减小(P<0.05或0.01)；但粉防已碱组和阳性对照组之间无差异(均P>0.05)。粉防已碱组、阳性对照组瘤重比模型组显著降低(P<0.05或0.01)；但粉防已碱组与阳性对照组之间无差异(P>0.05)。粉防已碱组和阳性对照组的抑瘤率分别为25.47%和30.43%。粉防已碱组、阳性对照组移植瘤组织病理学提示腺癌组织中可见坏死及不完全坏死细胞,凋亡现象比模型组增加,凋亡细胞较多。透射电镜观察发现粉防已碱组可见多量凋亡小体形成。与模型组比较,粉防已碱组和阳性对照组均能降低Bcl-2阳性细胞面积、光密度值和免疫组化指数(P<0.05或0.01)。与模型组比较,粉防已碱组和阳性对照组均能增高Bax阳性细胞面积和免疫组化指数(均P<0.01),粉防已碱组Bax光密度值与模型组比较无差异(P>0.05)。粉防已碱组、阳性对照组Survivin阳性细胞面积和免疫组化指数与模型组比较显著降低(均P<0.01),但粉防已碱组、阳性对照组Survivin光密度值与模型组比较无差异(均P>0.05)。与模型组比较,粉防已碱组和阳性对照组均能降低VEGF阳性细胞面积、光密度值和免疫组化指数(均P<0.01),粉防已碱组和阳性对照组之间比较差异无统计学意义(P>0.05)。4结论(1)首次发现粉防已碱对雌激素受体阴性乳腺癌细胞系MDA-MB-435S增殖有明显抑制作用,为临床用于雌激素受体阴性乳腺癌治疗方面提供了基础研究数据。(2)粉防已碱分别干预MDA-MB-435S细胞24h、48h后可诱导早期凋亡,早期凋亡率与药物浓度呈正相关,且存在浓度和时间依赖性,其最佳浓度范围为0.5μg/ml-4μg／ml。(3)粉防已碱阻滞MDA-MB-435S细胞于G0／G,期,降低S期比例,抑制DNA合成是其抑制乳腺癌细胞生长的机制之一(4)粉防已碱对MCF-7裸鼠移植瘤的生长有抑制作用。(5)粉防已碱抑制MCF-7裸鼠移植瘤生长的作用机制与下调Bcl-2、Survivin、上调Bax蛋白表达而诱导凋亡有关,同时与下调VEGF蛋白表达而抗肿瘤新生血管形成有关。更多还原

【Abstract】 1 ObjectiveBreast cancer is one of most common adult female malignant tumors. About 1/3 breast cancer patients show the estrogen receptor(ER) negative, which is insensitive to the chemotherapy and the endocrine therapy. Its malignant degree is higher and prognosis is worse than ER positive breast cancer. At present there are not ideal therapeutic methods. The treatment of ER negative breast cancer is the clinical difficulty. Therefore this research studies the antitumor effect and mechanism of tetrandrine(Tet) on ER negative human breast cancer cell MDA-MB-435S in Vitro and MCF-7 in tumor bearing nude mice,which is to lay the foundation for further studies in clinical treatments.2 Methods2.1 Experiments in VitroMTT was used to detect optical density (OD) of human breast cancer cell MDA-MB-435S by intervention of Tet in seven different concentration groups in 24h、48h、72h、96h. Drawing cell growth curve,calculating cell inhibitory rate and 50% inhibitory concentration (IC50) in every group, which were used to show inhibitory effect of Tet on human breast cancer cell MDA-MB-435S in Vitro growth.Propidium iodide (PI) single-staining flow cytometry assay method was used to analyze cell cycles of human breast cancer cell MDA-MB-435S by intervention of Tet in four different concentration groups. To observe cell percentage distribution in G0/G1, G2M, S phases by intervention of different Tet concentration groups after 24h.Cell apoptosis inspection method of Annexin V-FITC,PI double labelling was used to detect the cell apoptosis effect of Tet on human breast cancer cell MDA-MB-435S in four different concentration groups after 24h and 48h.2.2 Experiments in VivoTo build transplanted tumor animal model of human breast cancer cell MCF-7 in nude mice.24 mice with transplanted tumor were randomly divided into model group, Tet group and positive control group,8 mice in each group, administration for 15 days. Detecting and recording the growth condition and volumes of transplanted tumor in every group. Nude mice were killed in the day after administration ending up, weighting the transplanted tumor and calculating tumor inhibitory rate. Light microscope and transmission electron microscopy (TEM) were used to observe histopathologic changes and ultrastructural changes of transplanted tumors in every group. Image quantitative analysis was used to observe Bcl-2, Bax, Survivin and VEGF protein expression level of transplanted tumors in every group.3 Results3.1 Experiments in VitroAs compared with normal control group (control-1) at the same time, OD value was decreased significantly(P< 0.05 or 0.01)in seven different Tet concentration groups (Tet-0.5 group, Tet-1 group, Tet-2 group, Tet-4 group, Tet-6 group, Tet-8 group, Tet-16 group),and OD value decreased more significantly as Tet concentration increased. The inhibitory rate of Tet was enlarged with the increase of the concentration in the same time, which exhibited dose-effect relationship. With the passage of time, inhibitory effect of Tet on MDA-MB-435S cell in vitro growth was enlarged, which exhibited time-dependent effect. Interaction effect of dose and time existed for the effect of Tet on MDA-MB-435S cell (P<0.01). IC50 value in 24h,48h,72h,96h were 8.748μg/ml,3.585μg/ml, 1.451μg/ml,1.117μg/ml respectively.As compared with normal control group in the same phase, cell percentage of S phase, G2M phase in MDA-MB-435S cell were decreased significantly in four different Tet concentration groups (Tet-0.5 group, Tet-1 group, Tet-2 group, Tet-4 group) after administration for 24h, with statistical significance (P< 0.05).Higher the Tet concentration, lower the cell percentage of S phase, G2M phase. As compared with normal control group in the same phase, cell percentage of Go/Gi phase were increased significantly, with statistical significance (P < 0.05). Higher the Tet concentration, higher the cell percentage of G0/G1 phase.As compared with control-1 group after administration for 24h, early apoptosis rate was increased in four different Tet concentration groups (Tet-0.5 group, Tet-1 group, Tet-2 group, Tet-4 group),with statistical significance (P< 0.05). As compared with control-1 group after administration for 48h, early apoptosis rate was increased significantly in Tet-1 group, Tet-2 group and Tet-4 group (P< 0.05).Significant positive correlation was observed between early apoptosis rate and Tet concentration after administration for 24h,48h (P<0.05). With the passage of time, early apoptosis rate were increased gradually. There were no difference in late apoptosis rate between Tet groups and control-1 group after administration for 24h(P> 0.05). As compared with control-1 group after administration for 48h, late apoptosis rate were increased gradually in Tet high-concentration groups, with statistical significance (P< 0.05). 3.2 Experiments in VivoFrom 6 days after treatment, volumes of transplanted tumor in Tet group and positive control group were significantly smaller than model group (P<0.05 or 0.01), there were no difference between Tet group and positive control group (P> 0.05).The weight of the transplanted tumor in Tet group and positive control group were significantly decreased than model group (P<0.05 or 0.01) but there were no difference between Tet group and positive control group (P> 0.05). Tumor inhibitory rate in Tet group and positive control group were 25.47% and 30.43% respectively. Transplanted tumor in Tet group and positive control group by hemotoxylin and eosin staining suggested that necrotic cells and incomplete necrotic cells were seen among the adenocarcinoma. More apoptotic cells were found in Tet group and positive control group than model group. It was found by TEM that there were many apoptotic bodies in Tet group.As compared with model group, the positive cell area, mean optical density and immunohistochemistry index of Bcl-2 in Tet group and positive control group were significantly reduced (P<0.05 or 0.01). As compared with model group, the positive cell area and immunohistochemistry index of Bax in Tet group and positive control group were significantly increased (P<0.01). There were no difference of mean optical density between Tet group and positive control group (P>0.05). As compared with model group, the positive cell area and immunohistochemistry index of Survivin in Tet group and positive control group were significantly reduced (P<0.01). There were no difference of mean optical density among three groups (P>0.05). As compared with model group, the positive cell area, mean optical density and immunohistochemistry index of VEGF in Tet group and positive control group were significantly reduced (P<0.01),but There were no difference between Tet group and positive control group (P> 0.05).4 Conclusion(1) It is first found that the effect of Tet on inhibiting ER negative breast cancer MDA-MB-435S cell proliferation is obvious, which provides the basic research data for clinical treatments on ER negative breast cancer.(2) Tet can induce the early apoptosis after administration for 24h,48h. There is significant positive correlation between early apoptosis rate and Tet concentration.It has concentration and time dependence in the range of 0.5μg /ml to 4μg/ml.(3)Blocking the cell cycle at G0/G1 phase and reducing the cell percentage of S phase to inhibit the DNA synthesis is one of the antitumor mechanisms of Tet on breast cancer cell in Vitro.(4)Tet can inhibit the growth of transplanted tumor of MCF-7 in nude mice.(5)The antitumor mechanisms of Tet on MCF-7 in tumor bearing nude mice are related to reducing Bcl-2, Survivin, VEGF protein expression and increasing Bax protein expression.更多还原