低氧微环境中基底硬度对乳腺癌细胞MCF-7表型和上皮间质转化的影响
发布时间：2018-08-14 11:11
【摘要】：目的:本文通过模拟人乳腺癌恶性组织微环境,探究低氧和基底硬度共同作用对乳腺癌细胞MCF-7细胞形态、细胞活力和上皮间质转化(epithelial-mesenchymal transition,EMT)的影响。方法:制备硬度分别为0.5 kPa、5 kPa和20 kPa的聚丙烯酰胺凝胶,模拟恶性乳腺癌组织中的刚度范围。常氧和1%低氧微环境下在不同硬度基底上通过细胞骨架染色检测细胞形态的改变;利用live/dead染色及Hoechst染色检测细胞活力;利用Western blot和免疫荧光方法检测EMT上皮标志蛋白E-钙粘蛋白(E-cadherin)、间质标志蛋白波形蛋白(Vimentin)和转录因子Snail 1以及低氧诱导因子(hypoxia inducible factor,HIF?的表达;通过qPCR检测HIF-1?、E-cadherin、Vimentin、Snail1、基质金属蛋白酶2(matrix metalloproteinase 2,MMP 2)和基质金属蛋白酶9(matrix metalloproteinase 9,MMP 9)的基因表达。结果:常氧和1%低氧微环境下,不同硬度基底对MCF-7细胞的生物学行为具有重要影响。(1)细胞骨架染色结果表明:在常氧和1%低氧微环境中,不同硬度基底上,随着基底硬度的升高,细胞由圆形逐渐变成不规则多边形,细胞铺展面积增大,细胞圆度降低。(2)Live/dead染色和Hoechst染色结果表明:在常氧微环境中,不同硬度基底对细胞活力没有显著性影响;1%低氧环境下,细胞凋亡在20 kPa时显著性增高;相同硬度(20 kPa)基底上,1%低氧环境中细胞凋亡明显高于常氧环境。(3)Western blot和免疫荧光染色结果表明:在相同硬度基底上,1%低氧微环境促进MCF-7细胞HIF-1?蛋白水平的表达,降低上皮标志蛋白E-cadherin的表达,促进间质标志蛋白Vimentin和转录因子Snail 1的表达。在相同氧浓度环境中,基底硬度对E-cadherin和Vimentin表达的影响不明显,促进Snai 1蛋白水平的表达。(4)基因水平qPCR检测结果表明:在相同硬度基底上,1%低氧微环境促进HIF-1?的mRNA表达,降低上皮标志物E-cadherin的mRNA表达,促进间质标志物Vimentin和转录因子Snai 1的mRNA表达,促进MMP 2和MMP 9的mRNA表达。在1%低氧微环境中,随着基底硬度的升高,E-cadherin的m RNA表达没有显著性差异;转录因子Snail 1的mRNA表达随着基底硬度的升高而升高,MMP 2和MMP 9的mRNA表达随着基底硬度的升高而升高。结论:1%低氧微环境可促进乳腺癌细胞MCF-7发生EMT。1%低氧微环境中,较硬基底(20 kPa)可诱导MCF-7细胞凋亡,促进上皮样表型的丢失和间质样表型的获得,导致细胞EMT的发生。研究结果对于进一步探索低氧微环境中不同基底硬度影响细胞EMT的分子机制具有重要意义。
[Abstract]:Aim: to investigate the effects of hypoxia and basal hardness on the morphology, viability and epithelial-mesenchymal transition of breast cancer cell line MCF-7 by simulating the microenvironment of malignant tissue of human breast cancer. Methods: polyacrylamide gels with a hardness of 0.5 KPA 5 kPa and 20 kPa were prepared to simulate the stiffness range of malignant breast cancer tissues. Cell morphology was detected by cytoskeleton staining under normoxic and 1% hypoxia microenvironment, and cell viability was detected by live/dead staining and Hoechst staining. EMT epithelial marker Ecadherin (E-cadherin), interstitial marker vimentin (Vimentin), transcription factor Snail 1 and hypoxia inducible factor (hypoxia inducible factor 1 were detected by Western blot and immunofluorescence. QPCR was used to detect the gene expression of HIF-1 E-cadherin (Snail1, matrix metalloproteinase 2 (matrix metalloproteinase 2 mMP2) and matrix metalloproteinase 9 (MMP9). Results: in normoxic and 1% hypoxic microenvironments, different hardness substrates had an important effect on the biological behavior of MCF-7 cells. (1) the results of cytoskeleton staining showed that: in normoxic and 1% hypoxic microenvironments, different hardness substrates were obtained. With the increase of substrate hardness, cells gradually changed from round to irregular polygon, cell spreading area increased and cell roundness decreased. (2) the results of Live/dead staining and Hoechst staining showed that: in normoxic microenvironment, the cell spreading area increased and the cell roundness decreased. There was no significant effect on cell viability in different hardness substrates under 1% hypoxia, apoptosis increased significantly at 20 kPa. Apoptosis in 1% hypoxia environment of the same hardness (20 kPa) substrate was significantly higher than that in normoxic environment. (3) Western blot and immunofluorescence staining results showed that 1% hypoxia microenvironment on the same hardness substrate promoted HIF-1? Protein level decreased the expression of epithelial marker protein E-cadherin and promoted the expression of interstitial marker protein Vimentin and transcription factor Snail 1. The effect of substrate hardness on the expression of E-cadherin and Vimentin was not obvious in the same oxygen concentration environment, and promoted the expression of Snai 1 protein level. (4) the results of qPCR detection at gene level showed that 1% hypoxia microenvironment promoted HIF-1 protein expression on the same hardness substrate. MRNA expression, mRNA expression of epithelial marker E-cadherin, mRNA expression of interstitial marker Vimentin and transcription factor Snai 1, mRNA expression of MMP 2 and MMP 9 were decreased. In 1% hypoxia microenvironment, there was no significant difference in m RNA expression of E-cadherin with the increase of substrate hardness, while the mRNA expression of transcription factor Snail 1 increased with the increase of substrate hardness. The mRNA expression of MMP2 and MMP 9 increased with the increase of substrate hardness. Conclusion 1% hypoxia microenvironment can promote the occurrence of EMT. 1% hypoxia microenvironment in breast cancer cells. The harder substrate (20 kPa) can induce apoptosis of MCF-7 cells, promote the loss of epithelioid phenotypes and the acquisition of interstitial phenotypes, and lead to the occurrence of EMT. The results are of great significance for further exploring the molecular mechanism of the effect of different substrate hardness on cell EMT in hypoxic microenvironment.
【学位授予单位】：重庆大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R737.9
本文编号：2182664
[Abstract]:Aim: to investigate the effects of hypoxia and basal hardness on the morphology, viability and epithelial-mesenchymal transition of breast cancer cell line MCF-7 by simulating the microenvironment of malignant tissue of human breast cancer. Methods: polyacrylamide gels with a hardness of 0.5 KPA 5 kPa and 20 kPa were prepared to simulate the stiffness range of malignant breast cancer tissues. Cell morphology was detected by cytoskeleton staining under normoxic and 1% hypoxia microenvironment, and cell viability was detected by live/dead staining and Hoechst staining. EMT epithelial marker Ecadherin (E-cadherin), interstitial marker vimentin (Vimentin), transcription factor Snail 1 and hypoxia inducible factor (hypoxia inducible factor 1 were detected by Western blot and immunofluorescence. QPCR was used to detect the gene expression of HIF-1 E-cadherin (Snail1, matrix metalloproteinase 2 (matrix metalloproteinase 2 mMP2) and matrix metalloproteinase 9 (MMP9). Results: in normoxic and 1% hypoxic microenvironments, different hardness substrates had an important effect on the biological behavior of MCF-7 cells. (1) the results of cytoskeleton staining showed that: in normoxic and 1% hypoxic microenvironments, different hardness substrates were obtained. With the increase of substrate hardness, cells gradually changed from round to irregular polygon, cell spreading area increased and cell roundness decreased. (2) the results of Live/dead staining and Hoechst staining showed that: in normoxic microenvironment, the cell spreading area increased and the cell roundness decreased. There was no significant effect on cell viability in different hardness substrates under 1% hypoxia, apoptosis increased significantly at 20 kPa. Apoptosis in 1% hypoxia environment of the same hardness (20 kPa) substrate was significantly higher than that in normoxic environment. (3) Western blot and immunofluorescence staining results showed that 1% hypoxia microenvironment on the same hardness substrate promoted HIF-1? Protein level decreased the expression of epithelial marker protein E-cadherin and promoted the expression of interstitial marker protein Vimentin and transcription factor Snail 1. The effect of substrate hardness on the expression of E-cadherin and Vimentin was not obvious in the same oxygen concentration environment, and promoted the expression of Snai 1 protein level. (4) the results of qPCR detection at gene level showed that 1% hypoxia microenvironment promoted HIF-1 protein expression on the same hardness substrate. MRNA expression, mRNA expression of epithelial marker E-cadherin, mRNA expression of interstitial marker Vimentin and transcription factor Snai 1, mRNA expression of MMP 2 and MMP 9 were decreased. In 1% hypoxia microenvironment, there was no significant difference in m RNA expression of E-cadherin with the increase of substrate hardness, while the mRNA expression of transcription factor Snail 1 increased with the increase of substrate hardness. The mRNA expression of MMP2 and MMP 9 increased with the increase of substrate hardness. Conclusion 1% hypoxia microenvironment can promote the occurrence of EMT. 1% hypoxia microenvironment in breast cancer cells. The harder substrate (20 kPa) can induce apoptosis of MCF-7 cells, promote the loss of epithelioid phenotypes and the acquisition of interstitial phenotypes, and lead to the occurrence of EMT. The results are of great significance for further exploring the molecular mechanism of the effect of different substrate hardness on cell EMT in hypoxic microenvironment.
【学位授予单位】：重庆大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R737.9
【参考文献】
相关期刊论文 前3条
1 Zhao-ji LIU;Gregg L.SEMENZA;Hua-feng ZHANG;;低氧诱导因子与乳腺癌转移(英文)[J];Journal of Zhejiang University-Science B(Biomedicine & Biotechnology);2015年01期
2 张小梅;吕永钢;徐志玲;杨力;;基质力学对肿瘤发生发展及肿瘤细胞生物学行为影响的研究进展[J];生物化学与生物物理进展;2014年06期
3 王永兴;姜永光;罗勇;赵佳晖;陈雅童;韩毅力;林云华;;缺氧微环境下缺氧诱导因子-1α对前列腺癌细胞上皮间质转化的影响[J];首都医科大学学报;2014年03期
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