三氧化二砷联合衣霉素诱导人乳腺癌MDA-MB-231细胞凋亡作用研究
发布时间：2018-08-08 19:28
【摘要】：目的:探讨三氧化二砷(arsenic trioxide As2O3)联合衣霉素(Tunicamycin TM)诱导人三阴乳腺癌细胞MDA-MB-231凋亡效应及其机制。方法:以人三阴乳腺癌MDA-MB-231细胞株为模型,常规细胞培养。实验分别设As2O3组、TM组和二者联合用药组。As2O3组和TM组均分别设以下浓度[空白对照(DMEM)\对照(DMEM+NaOH/DMSO、0.1、0.5、1、10、20、40、60、80μmol/L)],分别诱导细胞24 h,用MTT法检测细胞增殖抑制率;根据细胞增殖抑制率,选择0.5μmol/LTM为固定浓度,联合不同浓度As203作为联合组作用细胞24h,再通过MTT法检测联合组细胞增殖抑制率。倒置显微镜下观察细胞形态学变化。流式细胞术检测细胞凋亡率及线粒体膜电位变化。Western Blot检测内质网应激(endoplasmic reticulum stress ERS)标志性蛋白葡萄糖调节蛋白78(glucose-regulated protein78,GRP78)的蛋白表达量及ERS凋亡相关蛋白Caspase4。根据 GRP78 蛋白表达选择 1μmol/L As203联合 0.5μmol/L TM 组,用内质网应激抑制剂四苯基丁酸(Four phenyl butyric acid 4-PBA)预处理细胞2小时,药物诱导细胞24小时后再观察对上述蛋白表达效应的影响。结果:1、MTT结果显示,As2O3组对细胞既有微促增殖又有促凋亡作用,在0.1、0.5、1μmol/IL对细胞有促增殖作用,10μmol/L后对细胞有抑增殖和促凋亡作用,与对照组相比均具有统计学意义(P0.05);TM组对乳腺癌细胞具有生长抑制作用,且随着药物剂量增大,增殖抑制率明显增高,与对照组相比具有统计学意义(P0.05);As2O3联合TM组诱导细胞,在0.1、1μmol/L浓度As2O3联合TM时,细胞的增长抑制率没有在两者的作用下相抵消,反而出现两者相互作用下,增殖抑制率明显增高,与同浓度的As203组比较有显著差异性(P0.05)。2、光镜下As2O3组、TM组及两药联合组中对照细胞状态良好,形态饱满呈细长梭形,密度较大,随着药物剂量增加,贴壁细胞生长密度减小,细胞体皱缩变圆,碎片明显增多,且有大量细胞漂浮。3、As2O3 在0.1μmol/L、1μmol/L、10μmol/L、20μmol/L、40μmol/L的浓度时作用乳腺癌细胞24小时后,细胞凋亡率分别为13.7%± 1.06、16.3%±1.22、21.9%±1.5、23.5%±1.33、50.8%±1.03,与对照组 12.0%±1.15 具有明显差异(P0.05);联合用药的凋亡率分别是20.5%±0.66、22.8%±0.46、23.6±0.95、43.3 ±0.74、51.4%±0.76,与对照组12.0%±1.15有明显差异,具有统计学意义(P0.05)。4、不同浓度As2O3联合0.5μmol/LTM诱导细胞时,细胞的线粒体膜电位显著下降,与对照组相比有显著差异,具有统计学意义(P0.05)。5、As2O3诱导乳腺癌细胞凋亡时GRP78表达无显著增加,与对照组相比无显著性差异(P＞0.05);TM诱导乳腺癌细胞ERS时可见GRP78表达明显上调,以12μmol/L TM诱导细胞ERS的GRP78表达量最高,与对照组相比差异具有统计学意义(P0.05);两药联合诱导细胞时,1 μμmol/L的As2O3联合0.5 μmol/L的TM时GRP78的表达量最高,与对照组相比差异具有统计学意义(P0.05)。6、TM诱导乳腺癌细胞发生ERS时能上调caspase4的表达,与对照组相比差异具有统计学意义(P0.05);As2O3联合TM诱导乳腺癌细胞时caspase4的表达无明显上调或下调,与对照组相比无显著性差异(p0.05)。7、用4-PBA预处理细胞2h后,TM诱导细胞24小时,可见caspase4蛋白表达的上调被阻断,与未经4-PBA预处理组相比差异均具有统计学意义(P0.05);用As2O3联合TM诱导细胞,可见caspase4蛋白表达无明显变化,与As203联合TM组相比不具有统计学意义(P＞0.05)。结论:1、As2O3联合TM诱导人乳腺癌MDA-MB-231细胞的凋亡率优于单药;2、1 μmol/L As2O3联合0.5μmol/L TM能协同促进MDA-MB-231细胞凋亡,TM可逆转As2O3的促增殖作用为抑增殖和促凋亡效应;3、As2O3联合TM诱导人乳腺癌MDA-MB-231细胞凋亡可能通过线粒体和内质网应激两种凋亡途径。
[Abstract]:Objective: To investigate the effect and mechanism of arsenic trioxide (arsenic trioxide As2O3) combined with ycomycin (Tunicamycin TM) induced MDA-MB-231 apoptosis in human breast cancer cells of three yin. Methods: the normal cell culture of human three yin breast cancer MDA-MB-231 cell lines was used as a model, and the experiments were set up in As2O3 group, TM group and two combination group.As2O3 group and TM group, respectively. The following concentrations [blank control (DMEM) control (DMEM+NaOH/DMSO, 0.1,0.5,1,10,20,40,60,80 mu mol/L)] were respectively established to induce cell 24 h, respectively, and the inhibition rate of cell proliferation was detected by MTT method. According to the inhibitory rate of cell proliferation, 0.5 mu mol/LTM was selected as the fixed concentration, and the combination of different concentration As203 as the combined group of 24h, and then detected by MTT method. Cell proliferation inhibition rate and cell morphological changes under inverted microscope. Cell apoptosis rate and mitochondrial membrane potential change were detected by flow cytometry..Western Blot was used to detect the protein expression and ERS of endoplasmic reticulum stress (endoplasmic reticulum stress ERS) glucose regulator protein 78 (glucose-regulated protein78, GRP78) Apoptosis related protein Caspase4. was selected according to the expression of GRP78 protein, 1 u mol/L As203 combined with 0.5 mol/L TM group, and the cells were pretreated with endoplasmic reticulum stress inhibitor four phenyl butyric acid (Four phenyl butyric acid 4-PBA) for 2 hours. The effect of the drug induced cells after 24 hours was observed. Results: 1, MTT results showed that The 3 groups had both micro proliferation and apoptotic effect on cells. The cell proliferation was promoted by 0.1,0.5,1 mu mol/IL. After 10 mol/L, the cells had inhibitory proliferation and apoptosis effect. Compared with the control group, the cells had statistical significance (P0.05). The TM group had inhibitory effect on the growth of breast cancer cells, and with the increase of drug dosage, the proliferation inhibition rate was clear. The increase was statistically significant (P0.05) compared with the control group (P0.05). The inhibition rate of cell growth was not counteracted under the action of both 0.1,1 mu mol/L and As2O3 combined with TM in the group of 0.1,1 mu mol/L, but the proliferation inhibition rate increased significantly under the interaction of the two groups (P0.0), and there was a significant difference compared with the same concentration of As203 group (P0.0). 5).2, the control cells in group As2O3, TM group and two drug group under light microscope were in good condition, the shape was long spindle shape, and the density was large. With the increase of drug dosage, the growth density of the adherent cells decreased, the cell body crinkled and turned round, the fragments increased obviously, and there were a large number of cells floating.3, As2O3 was 0.1 mu mol/L, 1 mu mol/L, 10 u mol/L, 20 u mol/L, 40 micron mol/L. The apoptosis rate of breast cancer cells was 13.7% + 1.06,16.3% + 1.5,23.5% + 1.5,23.5% + 1.33,50.8% + 1.03 respectively, which was significantly different from that of the control group (P0.05), respectively (P0.05), and the rate of apoptosis was 20.5% + 0.66,22.8% + 0.66,22.8% + 0.95,43.3 + 0.74,51.4% + 0.76, respectively, and 12% + 1.15 with the control group. The significant difference was statistically significant (P0.05).4. The mitochondrial membrane potential of the cells decreased significantly when different concentrations of As2O3 combined with 0.5 u mol/LTM, compared with the control group, with statistical significance (P0.05).5. There was no significant increase in GRP78 expression when As2O3 induced the apoptosis of breast cancer cells, and there was no significant difference compared with the control group. P > 0.05); when TM induced ERS in breast cancer cells, the expression of GRP78 was obviously up-regulated, and the expression of GRP78 in cell ERS was highest with 12 mu mol/L TM, and the difference was statistically significant compared with the control group (P0.05). When the two drugs were combined to induce cells, the highest expression of the 1 mu mol/L As2O3 combined 0.5 micron mol/L was compared with the control group. There was statistically significant (P0.05).6, TM induced the expression of caspase4 in breast cancer cells induced by ERS, and the difference was statistically significant compared with the control group (P0.05). As2O3 combined TM induced breast cancer cells without significant up-regulation or down regulation, and there was no significant difference compared with the control group (P0.05).7. The up - regulation of caspase4 protein expression was blocked for 24 hours, and the difference was statistically significant compared with that of the 4-PBA preconditioning group (P0.05). There was no significant change in the expression of caspase4 protein by As2O3 combined with TM. Compared with the As203 combined TM group, there was no statistical significance (P > 0.05). Conclusion: 1, As2O3 combined TM inducer. The apoptosis rate of MDA-MB-231 cells in breast cancer is better than that of single drug; 2,1 mu mol/L As2O3 combined with 0.5 mu mol/L TM can promote apoptosis of MDA-MB-231 cells, TM can reverse the proliferation promoting effect of As2O3 to inhibit proliferation and promote apoptosis effect. 3, As2O3 combined TM induces apoptosis in human breast cancer MDA-MB-231 cells by two kinds of apoptotic pathways through mitochondria and endoplasmic reticulum stress. Diameter.
【学位授予单位】：贵州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R96
本文编号：2172817
[Abstract]:Objective: To investigate the effect and mechanism of arsenic trioxide (arsenic trioxide As2O3) combined with ycomycin (Tunicamycin TM) induced MDA-MB-231 apoptosis in human breast cancer cells of three yin. Methods: the normal cell culture of human three yin breast cancer MDA-MB-231 cell lines was used as a model, and the experiments were set up in As2O3 group, TM group and two combination group.As2O3 group and TM group, respectively. The following concentrations [blank control (DMEM) control (DMEM+NaOH/DMSO, 0.1,0.5,1,10,20,40,60,80 mu mol/L)] were respectively established to induce cell 24 h, respectively, and the inhibition rate of cell proliferation was detected by MTT method. According to the inhibitory rate of cell proliferation, 0.5 mu mol/LTM was selected as the fixed concentration, and the combination of different concentration As203 as the combined group of 24h, and then detected by MTT method. Cell proliferation inhibition rate and cell morphological changes under inverted microscope. Cell apoptosis rate and mitochondrial membrane potential change were detected by flow cytometry..Western Blot was used to detect the protein expression and ERS of endoplasmic reticulum stress (endoplasmic reticulum stress ERS) glucose regulator protein 78 (glucose-regulated protein78, GRP78) Apoptosis related protein Caspase4. was selected according to the expression of GRP78 protein, 1 u mol/L As203 combined with 0.5 mol/L TM group, and the cells were pretreated with endoplasmic reticulum stress inhibitor four phenyl butyric acid (Four phenyl butyric acid 4-PBA) for 2 hours. The effect of the drug induced cells after 24 hours was observed. Results: 1, MTT results showed that The 3 groups had both micro proliferation and apoptotic effect on cells. The cell proliferation was promoted by 0.1,0.5,1 mu mol/IL. After 10 mol/L, the cells had inhibitory proliferation and apoptosis effect. Compared with the control group, the cells had statistical significance (P0.05). The TM group had inhibitory effect on the growth of breast cancer cells, and with the increase of drug dosage, the proliferation inhibition rate was clear. The increase was statistically significant (P0.05) compared with the control group (P0.05). The inhibition rate of cell growth was not counteracted under the action of both 0.1,1 mu mol/L and As2O3 combined with TM in the group of 0.1,1 mu mol/L, but the proliferation inhibition rate increased significantly under the interaction of the two groups (P0.0), and there was a significant difference compared with the same concentration of As203 group (P0.0). 5).2, the control cells in group As2O3, TM group and two drug group under light microscope were in good condition, the shape was long spindle shape, and the density was large. With the increase of drug dosage, the growth density of the adherent cells decreased, the cell body crinkled and turned round, the fragments increased obviously, and there were a large number of cells floating.3, As2O3 was 0.1 mu mol/L, 1 mu mol/L, 10 u mol/L, 20 u mol/L, 40 micron mol/L. The apoptosis rate of breast cancer cells was 13.7% + 1.06,16.3% + 1.5,23.5% + 1.5,23.5% + 1.33,50.8% + 1.03 respectively, which was significantly different from that of the control group (P0.05), respectively (P0.05), and the rate of apoptosis was 20.5% + 0.66,22.8% + 0.66,22.8% + 0.95,43.3 + 0.74,51.4% + 0.76, respectively, and 12% + 1.15 with the control group. The significant difference was statistically significant (P0.05).4. The mitochondrial membrane potential of the cells decreased significantly when different concentrations of As2O3 combined with 0.5 u mol/LTM, compared with the control group, with statistical significance (P0.05).5. There was no significant increase in GRP78 expression when As2O3 induced the apoptosis of breast cancer cells, and there was no significant difference compared with the control group. P > 0.05); when TM induced ERS in breast cancer cells, the expression of GRP78 was obviously up-regulated, and the expression of GRP78 in cell ERS was highest with 12 mu mol/L TM, and the difference was statistically significant compared with the control group (P0.05). When the two drugs were combined to induce cells, the highest expression of the 1 mu mol/L As2O3 combined 0.5 micron mol/L was compared with the control group. There was statistically significant (P0.05).6, TM induced the expression of caspase4 in breast cancer cells induced by ERS, and the difference was statistically significant compared with the control group (P0.05). As2O3 combined TM induced breast cancer cells without significant up-regulation or down regulation, and there was no significant difference compared with the control group (P0.05).7. The up - regulation of caspase4 protein expression was blocked for 24 hours, and the difference was statistically significant compared with that of the 4-PBA preconditioning group (P0.05). There was no significant change in the expression of caspase4 protein by As2O3 combined with TM. Compared with the As203 combined TM group, there was no statistical significance (P > 0.05). Conclusion: 1, As2O3 combined TM inducer. The apoptosis rate of MDA-MB-231 cells in breast cancer is better than that of single drug; 2,1 mu mol/L As2O3 combined with 0.5 mu mol/L TM can promote apoptosis of MDA-MB-231 cells, TM can reverse the proliferation promoting effect of As2O3 to inhibit proliferation and promote apoptosis effect. 3, As2O3 combined TM induces apoptosis in human breast cancer MDA-MB-231 cells by two kinds of apoptotic pathways through mitochondria and endoplasmic reticulum stress. Diameter.
【学位授予单位】：贵州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R96
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