三氧化二砷对人雌激素受体阴性乳腺癌细胞株作用的研究

【作者】 张琪；

【导师】 裴晓华；

【作者基本信息】 北京中医药大学 ， 中医外科学， 2009， 硕士

【摘要】 乳腺癌是全世界妇女中发病率最高的恶性肿瘤,也是临床上最常见的激素依赖性肿瘤。根据ER表型差异,乳腺癌被分为两种:ER阳性乳腺癌和ER阴性乳腺癌。大约2/3乳腺癌患者呈ER阳性,肿瘤细胞生长具有雌激素依赖性,对于这类患者,临床上常采取撤除雌激素的内分泌治疗方法。大约1/3乳腺癌患者为ER阴性,与ER阳性乳腺癌相比,其恶性度较高,对内分泌治疗不敏感,目前尚无理想的治疗方法,预后较差。若能诱导ER阴性乳腺癌细胞凋亡,将为ER阴性乳腺癌患者的治疗开辟新的治疗途径,为这些患者带来希望。三氧化二砷（分子式As₂O₃）为无机化合物,是中药砒霜的提取物,有剧毒。长期以来As₂O₃的药用价值被其剧毒性所掩盖。现在随着质控技术的发达和对As₂O₃实验及临床研究的深入,As₂O₃在肿瘤治疗方面的临床应用范围越来越广泛。现代药理研究说明,三氧化二砷对白血病和许多实体瘤有不同程度的抑制作用,中医文献也有不少有关砒霜治疗肿瘤的记载。以此为依据,本实验以MDA-MB-435S细胞为研究对象,研究三氧化二砷对雌激素受体阴性乳腺癌细胞株的细胞生长、细胞形态、细胞周期、细胞凋亡及凋亡相关基因的影响情况,探讨三氧化二砷对ER阴性乳腺癌细胞株的作用机制,在中医治疗ER阴性乳腺癌方面开辟了新思路。【目的】以雌激素受体阴性乳腺癌细胞株MDA-MB-435S为研究对象,研究三氧化二砷对MDA-MB-435S细胞增殖、细胞周期、凋亡的影响,并研究其可能的作用机制。【方法】实验分六部分:MTT实验、光镜观察细胞形态、流式PI单染检测细胞周期和凋亡、流式Annexin V/PI双染检测细胞凋亡、免疫组化检测细胞内Bcl-2和Bax表达情况、PI 33342荧光染色观察细胞凋亡。在体外培养雌激素受体阴性乳腺癌细胞株MDA-MB-435S,采用不同浓度连续作用于细胞。通过MTT比色实验,观察不同施加因素对细胞生长的影响,并根据MTT实验结果初步筛选三氧化二砷的作用浓度,在量效曲线1.75-28μg/ml的线性关系范围内,在IC₅₀值为10.47μg/mL上下选取三氧化二砷的加药浓度,设对照组和加药组（1、5、10、15、20μg/ml）进行流式细胞仪技术检测细胞周期及凋亡的变化,根据流式检测结果选取对照组和加药组（0.25、0.5、1、5、10μg/ml）进行免疫组化检测Bcl-2和Bax的变化,并通过PI 33342荧光染色实验观察细胞凋亡情况。【结果】1.大于1.75μg/ml的三氧化二砷能明显抑制MDA-MB-435S细胞生长,在1.75～28μg/ml浓度范围内抑制作用明显（P＜0.001）,且有剂量依赖效应。2.三氧化二砷可以使MDA-MB-435S细胞周期中的G0-G1期细胞比率增高,G2/M期细胞比率增高,S期细胞比率下降,与对照组比,结果均有显著性差异（p＜0.05）。3.流式PI单染结果显示:1、5、10、15、20μg/ml浓度范围内,MDA-MB-435S细胞未见明显凋亡,而MCF-7细胞可见明显凋亡（p＜0.05）。4.Annexin V/PI双染结果显示:1、5、10、15、20μg/ml浓度与对照组相比,MDA-MB-435S细胞的早期凋亡率未见显著性差异;0.25、0.5、1、1.5、2μg/ml浓度与对照组相比,MDA-MB-435S细胞的早期凋亡率均有显著性差异（p＜0.05）,且随加药浓度的增加凋亡率明显增高。说明低浓度三氧化二砷可能诱导MDA-MB-435S细胞凋亡。5.免疫组化结果显示:三氧化二砷组（0.25、0.5、1、5、10μg/ml）与对照组相比,可以下调BCL-2表达和BAX表达。6.PI 33342荧光染色结果显示:相同条件下,三氧化二砷组（0.25、0.5、1、5、10μg/ml）与空白对照组相比,MDA-MB-435S细胞未见明显凋亡细胞形态,MCF-7细胞可见凋亡细胞形态。【结论】1.三氧化二砷作用于MDA-MB-435S细胞可抑制细胞增殖。2.三氧化二砷可阻滞MDA-MB-435S细胞周期在G0-G1和G2-M期,使进入S期细胞减少,可能诱导MDA-MB-435S细胞凋亡。3.三氧化二砷可以下调BCL-2表达和BAX表达,三氧化二砷可能是通过下调BCL-2表达的方式来达到抑制MDA-MB-435S细胞生长的作用,但三氧化二砷对MDA-MB-435S细胞抑制作用的方式有待进一步研究。更多还原

【Abstract】 Women breast cancer is the world’s highest incidence of malignant tumor,and is clinically the most common hormone-dependent tumor. According to phenotypic differences in ER breast cancer were divided into two types:ER-positive breast cancer and ER-negative breast cancer.About 2 / 3 were ER-positive breast cancer patients,tumor cell growth with estrogen-dependent.For such patients,clinicians often take estrogen removal of endocrine treatment.Approximately 1 / 3 for ER-negative breast cancer patients with ER-positive breast cancer compared to a higher grade, is not sensitive to endocrine therapy,there is no ideal method of treatment, poor prognosis.If the induction of apoptosis in ER-negative breast cancer will be ER-negative breast cancer patients to open up new therapeutic approaches,in order to bring hope to these patients.As₂O₃ which is inorganic compounds,is Chinese medicine extract arsenic, having Highly toxic.A long period of time,the medicinal value of As₂O₃ has been overshadowed by its highly toxic.Now,with advanced quality control technology and deep studies on experimental and clinical of As₂O₃,its treatment of Cancer Clinical application is more and more wide. Modern pharmacological studies indicated that,As₂O₃ has varying degrees of inhibition for leukemia and many solid tumors.Chinese medicine also has a lot of literature on the arsenic treatment of documented tumor. In the experiment,we study that arsenic trioxide roles in estrogen receptor-negative breast cancer cell lines MDA-MB-435S of cell growth, cell morphology,cell cycle,apoptosis and apoptosis-related gene effect.We investigate the effect of arsenic trioxide on the ER-negative breast cancer cell lines the role of mechanisms of Chinese medicine treatment in ER-negative breast cancer has opened up new ideas.ObjectiveTo investigate the influence of arsenic on the cell proliferation, mitotic cycle and apoptosis of MDA-MB-435S and discuss the mechanism.MethodThe experiment was divided into six parts:MTT experiment,Light microscope to observe cell morphology,PI-flow single-dye experiments to measure the changes of cell cycle and apoptosis,Streaming AnnexinⅤ/ PI double staining to measure cell apoptosis,Immunohistochemical detection of intracellular Bcl-2 and Bax expression,PI 33342 fluorescent staining of apoptosis.We adopt the role of different concentrations in vitro MDA-MB-435S.The influence of different factors on growth of the cells were observed by MTT colorimetry assay and corresponding densities of As₂O₃ were screened preliminary by the results.At 1.75-28μg/ml range of linear relationship by dose-response curves,we select corresponding densities of As₂O₃ at upper and lower of IC50 value.Located the control group and the As₂O₃ group（1、5、10、15、20μg/ml）,we measure the changes of cell cycle and apoptosis by flow cytometry,according to flow test results,we selecte the control group and the As₂O₃ group（1、5、10、15、20μg/ml） for being detected BCL-2 and BAX changes by immunohistochemical method.Cell apoptosis were observed by PI 33342 fluorescence staining experiments.results1.Greater than 1.75μg/ml arsenic trioxide could significantly inhibit the growth of MDA-MB-435S cells,and arsenic trioxide have a dose effect in 1.75～28μg/ml concentration range（P<0.001）.2.Arsenic Trioxide can make the percentage of G0-G1 stage cells and G2/M stage cells increase in the MDA-MB-435S cell cycle,and make S stage cell decrease.To contrast with control group,all of the results have significant differences（p<0.05）.3.PI-flow single-dye experiments show that MDA-MB-435S cells do not have obvious apoptosis and MCF-7 cells have obvious apoptosis in 1、5、10、15、20μg/ml（p<0.05）.4.Streaming AnnexinⅤ/ PI double staining experiments show that the early apoptosis rate of MDA-MB-435S cells have no significant difference by As₂O₃ group（1、5、10、15、20μg/ml）compared with control group,and the early apoptosis rate of MDA-MB-435S cells have significant difference by As₂O₃ group（0.25、0.5、1、1.5、2μg/ml） compared with control group.With the action dose heightening,the rate of Apoptosis will be induced to increase obviously.MDA-MB-435S cells apoptosis probably induced by low concentrations of arsenic trioxide.5.Immunohistochemical detection experiments show that the group of Arsenic Trioxide（0.25、0.5、1、5、10μg/ml） compared with control group can down-regulate the express of BCL-2 and BAX.6.PI 33342 fluorescent staining experiments show that,under the same conditions,the group of Arsenic Trioxide（0.25、0.5、1、5、10μg/ml ）compared with control group,apoptotic cells morphology can not be seen obviously in MDA-MB-435S cells and can be seen in MCF-7 cells.conclusion1.arsenic trioxide can inhibit MDA-MB-435S cell proliferation.2.Arsenic trioxide can block MDA-MB-435S cell cycle at G0-G1 and G2-M phase,so that reduction into the S phase cells,probably induced by MDA-MB-435S cell apoptosis.3.Arsenic Trioxide can down-regulate the express of BCL-2 and BAX. Arsenic trioxide are possible through reduced expression of BCL-2 to attain the inhibition MDA-MB-435S cells.Inhibition of the way should be studied further.更多还原