低氧微環(huán)境中基底硬度對乳腺癌細胞MCF-7表型和上皮間質轉化的影響
發(fā)布時間：2018-08-14 11:11
【摘要】：目的:本文通過模擬人乳腺癌惡性組織微環(huán)境,探究低氧和基底硬度共同作用對乳腺癌細胞MCF-7細胞形態(tài)、細胞活力和上皮間質轉化(epithelial-mesenchymal transition,EMT)的影響。方法:制備硬度分別為0.5 kPa、5 kPa和20 kPa的聚丙烯酰胺凝膠,模擬惡性乳腺癌組織中的剛度范圍。常氧和1%低氧微環(huán)境下在不同硬度基底上通過細胞骨架染色檢測細胞形態(tài)的改變;利用live/dead染色及Hoechst染色檢測細胞活力;利用Western blot和免疫熒光方法檢測EMT上皮標志蛋白E-鈣粘蛋白(E-cadherin)、間質標志蛋白波形蛋白(Vimentin)和轉錄因子Snail 1以及低氧誘導因子(hypoxia inducible factor,HIF?的表達;通過qPCR檢測HIF-1?、E-cadherin、Vimentin、Snail1、基質金屬蛋白酶2(matrix metalloproteinase 2,MMP 2)和基質金屬蛋白酶9(matrix metalloproteinase 9,MMP 9)的基因表達。結果:常氧和1%低氧微環(huán)境下,不同硬度基底對MCF-7細胞的生物學行為具有重要影響。(1)細胞骨架染色結果表明:在常氧和1%低氧微環(huán)境中,不同硬度基底上,隨著基底硬度的升高,細胞由圓形逐漸變成不規(guī)則多邊形,細胞鋪展面積增大,細胞圓度降低。(2)Live/dead染色和Hoechst染色結果表明:在常氧微環(huán)境中,不同硬度基底對細胞活力沒有顯著性影響;1%低氧環(huán)境下,細胞凋亡在20 kPa時顯著性增高;相同硬度(20 kPa)基底上,1%低氧環(huán)境中細胞凋亡明顯高于常氧環(huán)境。(3)Western blot和免疫熒光染色結果表明:在相同硬度基底上,1%低氧微環(huán)境促進MCF-7細胞HIF-1?蛋白水平的表達,降低上皮標志蛋白E-cadherin的表達,促進間質標志蛋白Vimentin和轉錄因子Snail 1的表達。在相同氧濃度環(huán)境中,基底硬度對E-cadherin和Vimentin表達的影響不明顯,促進Snai 1蛋白水平的表達。(4)基因水平qPCR檢測結果表明:在相同硬度基底上,1%低氧微環(huán)境促進HIF-1?的mRNA表達,降低上皮標志物E-cadherin的mRNA表達,促進間質標志物Vimentin和轉錄因子Snai 1的mRNA表達,促進MMP 2和MMP 9的mRNA表達。在1%低氧微環(huán)境中,隨著基底硬度的升高,E-cadherin的m RNA表達沒有顯著性差異;轉錄因子Snail 1的mRNA表達隨著基底硬度的升高而升高,MMP 2和MMP 9的mRNA表達隨著基底硬度的升高而升高。結論:1%低氧微環(huán)境可促進乳腺癌細胞MCF-7發(fā)生EMT。1%低氧微環(huán)境中,較硬基底(20 kPa)可誘導MCF-7細胞凋亡,促進上皮樣表型的丟失和間質樣表型的獲得,導致細胞EMT的發(fā)生。研究結果對于進一步探索低氧微環(huán)境中不同基底硬度影響細胞EMT的分子機制具有重要意義。
[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.
【學位授予單位】：重慶大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R737.9
本文編號：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.
【學位授予單位】：重慶大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R737.9
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