三氧化二砷聯(lián)合衣霉素誘導(dǎo)人乳腺癌MDA-MB-231細胞凋亡作用研究
發(fā)布時間：2018-08-08 19:28
【摘要】：目的:探討三氧化二砷(arsenic trioxide As2O3)聯(lián)合衣霉素(Tunicamycin TM)誘導(dǎo)人三陰乳腺癌細胞MDA-MB-231凋亡效應(yīng)及其機制。方法:以人三陰乳腺癌MDA-MB-231細胞株為模型,常規(guī)細胞培養(yǎng)。實驗分別設(shè)As2O3組、TM組和二者聯(lián)合用藥組。As2O3組和TM組均分別設(shè)以下濃度[空白對照(DMEM)\對照(DMEM+NaOH/DMSO、0.1、0.5、1、10、20、40、60、80μmol/L)],分別誘導(dǎo)細胞24 h,用MTT法檢測細胞增殖抑制率;根據(jù)細胞增殖抑制率,選擇0.5μmol/LTM為固定濃度,聯(lián)合不同濃度As203作為聯(lián)合組作用細胞24h,再通過MTT法檢測聯(lián)合組細胞增殖抑制率。倒置顯微鏡下觀察細胞形態(tài)學變化。流式細胞術(shù)檢測細胞凋亡率及線粒體膜電位變化。Western Blot檢測內(nèi)質(zhì)網(wǎng)應(yīng)激(endoplasmic reticulum stress ERS)標志性蛋白葡萄糖調(diào)節(jié)蛋白78(glucose-regulated protein78,GRP78)的蛋白表達量及ERS凋亡相關(guān)蛋白Caspase4。根據(jù) GRP78 蛋白表達選擇 1μmol/L As203聯(lián)合 0.5μmol/L TM 組,用內(nèi)質(zhì)網(wǎng)應(yīng)激抑制劑四苯基丁酸(Four phenyl butyric acid 4-PBA)預(yù)處理細胞2小時,藥物誘導(dǎo)細胞24小時后再觀察對上述蛋白表達效應(yīng)的影響。結(jié)果:1、MTT結(jié)果顯示,As2O3組對細胞既有微促增殖又有促凋亡作用,在0.1、0.5、1μmol/IL對細胞有促增殖作用,10μmol/L后對細胞有抑增殖和促凋亡作用,與對照組相比均具有統(tǒng)計學意義(P0.05);TM組對乳腺癌細胞具有生長抑制作用,且隨著藥物劑量增大,增殖抑制率明顯增高,與對照組相比具有統(tǒng)計學意義(P0.05);As2O3聯(lián)合TM組誘導(dǎo)細胞,在0.1、1μmol/L濃度As2O3聯(lián)合TM時,細胞的增長抑制率沒有在兩者的作用下相抵消,反而出現(xiàn)兩者相互作用下,增殖抑制率明顯增高,與同濃度的As203組比較有顯著差異性(P0.05)。2、光鏡下As2O3組、TM組及兩藥聯(lián)合組中對照細胞狀態(tài)良好,形態(tài)飽滿呈細長梭形,密度較大,隨著藥物劑量增加,貼壁細胞生長密度減小,細胞體皺縮變圓,碎片明顯增多,且有大量細胞漂浮。3、As2O3 在0.1μmol/L、1μmol/L、10μmol/L、20μmol/L、40μmol/L的濃度時作用乳腺癌細胞24小時后,細胞凋亡率分別為13.7%± 1.06、16.3%±1.22、21.9%±1.5、23.5%±1.33、50.8%±1.03,與對照組 12.0%±1.15 具有明顯差異(P0.05);聯(lián)合用藥的凋亡率分別是20.5%±0.66、22.8%±0.46、23.6±0.95、43.3 ±0.74、51.4%±0.76,與對照組12.0%±1.15有明顯差異,具有統(tǒng)計學意義(P0.05)。4、不同濃度As2O3聯(lián)合0.5μmol/LTM誘導(dǎo)細胞時,細胞的線粒體膜電位顯著下降,與對照組相比有顯著差異,具有統(tǒng)計學意義(P0.05)。5、As2O3誘導(dǎo)乳腺癌細胞凋亡時GRP78表達無顯著增加,與對照組相比無顯著性差異(P＞0.05);TM誘導(dǎo)乳腺癌細胞ERS時可見GRP78表達明顯上調(diào),以12μmol/L TM誘導(dǎo)細胞ERS的GRP78表達量最高,與對照組相比差異具有統(tǒng)計學意義(P0.05);兩藥聯(lián)合誘導(dǎo)細胞時,1 μμmol/L的As2O3聯(lián)合0.5 μmol/L的TM時GRP78的表達量最高,與對照組相比差異具有統(tǒng)計學意義(P0.05)。6、TM誘導(dǎo)乳腺癌細胞發(fā)生ERS時能上調(diào)caspase4的表達,與對照組相比差異具有統(tǒng)計學意義(P0.05);As2O3聯(lián)合TM誘導(dǎo)乳腺癌細胞時caspase4的表達無明顯上調(diào)或下調(diào),與對照組相比無顯著性差異(p0.05)。7、用4-PBA預(yù)處理細胞2h后,TM誘導(dǎo)細胞24小時,可見caspase4蛋白表達的上調(diào)被阻斷,與未經(jīng)4-PBA預(yù)處理組相比差異均具有統(tǒng)計學意義(P0.05);用As2O3聯(lián)合TM誘導(dǎo)細胞,可見caspase4蛋白表達無明顯變化,與As203聯(lián)合TM組相比不具有統(tǒng)計學意義(P＞0.05)。結(jié)論:1、As2O3聯(lián)合TM誘導(dǎo)人乳腺癌MDA-MB-231細胞的凋亡率優(yōu)于單藥;2、1 μmol/L As2O3聯(lián)合0.5μmol/L TM能協(xié)同促進MDA-MB-231細胞凋亡,TM可逆轉(zhuǎn)As2O3的促增殖作用為抑增殖和促凋亡效應(yīng);3、As2O3聯(lián)合TM誘導(dǎo)人乳腺癌MDA-MB-231細胞凋亡可能通過線粒體和內(nèi)質(zhì)網(wǎng)應(yīng)激兩種凋亡途徑。
[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.
【學位授予單位】：貴州醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R96
本文編號：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.
【學位授予單位】：貴州醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R96
【參考文獻】
相關(guān)期刊論文 前10條
1 劉楚羽;謝明均;;三陰乳腺癌臨床靶向治療進展[J];重慶醫(yī)學;2017年02期
2 秦奕男;李丹丹;朱筑霞;;內(nèi)質(zhì)網(wǎng)應(yīng)激誘導(dǎo)人乳腺癌MDA-MB-231細胞系凋亡[J];基礎(chǔ)醫(yī)學與臨床;2016年07期
3 李靜;周偉強;;SAHA、TRAIL與乳腺癌細胞凋亡[J];沈陽醫(yī)學院學報;2016年03期
4 管宇;劉炳彤;臧韻;柳明洙;;珍珠梅黃酮納米粒通過活化內(nèi)質(zhì)網(wǎng)應(yīng)激的Caspase通路抑制大鼠原發(fā)性肝癌的生長[J];時珍國醫(yī)國藥;2015年07期
5 王新;范立君;李壯;王博;周昱;;GRP78、GRP94在乳腺癌中的表達及其意義[J];黑龍江醫(yī)藥科學;2014年06期
6 張維民;楊希才;;三氧化二砷抗腫瘤臨床運用進展[J];中外醫(yī)療;2014年26期
7 李索妮;姚煜;南克俊;;三陰性乳腺癌治療進展[J];現(xiàn)代腫瘤醫(yī)學;2014年01期
8 俞罡;;三氧化二砷治療復(fù)發(fā)性急性早幼粒細胞白血病的療效分析[J];中國醫(yī)藥指南;2013年22期
9 張凱強;張穎;顧何鋒;劉璐;馬林;;內(nèi)質(zhì)網(wǎng)應(yīng)激與細胞凋亡研究進展[J];口腔醫(yī)學;2013年06期
10 王靜霞;張秋榮;;三氧化二砷聯(lián)合沙利度胺對骨髓增生異常綜合征的調(diào)節(jié)作用[J];實用臨床醫(yī)藥雜志;2013年11期
相關(guān)博士學位論文 前1條
1 潘春;Bmbuffy基因在家蠶細胞內(nèi)源性線粒體凋亡途徑與細胞自噬中的功能研究[D];西南大學;2014年
相關(guān)碩士學位論文 前1條
1 胡磊;三氧化二砷對三陰性乳腺癌細胞（MDA-MB-231）的化療增敏作用[D];復(fù)旦大學;2012年
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