Aurora激酶抑制剂VX-680对食管癌细胞增殖、粘附、迁移及失巢凋亡的影响

发布时间：2018-08-05 16:46

【摘要】：目的:观察Aurora激酶抑制剂VX-680对食管癌KYSE150细胞增殖、凋亡、粘附、迁移及失巢凋亡的影响,并探讨其分子机制。方法:1.采用MTT法、DAPI染色法、细胞间粘附实验、细胞-细胞外基质粘附实验、划痕实验观察不同浓度(0μΜ、0.5μΜ、1μΜ)的VX-680对KYSE150细胞增殖、凋亡、失巢凋亡、粘附及迁移能力的影响;2.采用Real-time PCR技术检测粘附分子CD44和基质金属蛋白酶MMP-2的表达;3.采用Western blot技术检测不同浓度的VX-680作用KYSE150细胞后凋亡分子Caspase3、PARP,粘附分子E-cadherin、CD44及影响迁移的基质金属蛋白酶MMP-2分子蛋白表达情况;4.采用Western blot技术检测不同浓度的VX-680作用后对ERK/p-ERK、AKT/p-AKT蛋白表达水平的影响。结果:1.不同浓度的VX-680作用于食管癌细胞株KYSE150后,发现随着VX-680浓度的增加:MTT法实验结果显示细胞的增殖率逐渐降低(P0.05);DAPI染色法显示,细胞发生核浓缩、核碎裂的比例逐渐增高(P0.001);细胞间粘附实验显示,细胞聚集形成的团块更大,更紧密(P0.001);细胞间更难分离,离散程度逐渐减弱(P0.001);细胞-细胞外基质粘附实验显示,细胞与三种不同的外基质的粘附能力均逐渐减弱(P0.05);划痕实验结果显示,细胞的愈合能力逐渐减弱(P0.001)。2.Real-time PCR结果显示,随着VX-680浓度的增加,CD44和MMP-2的表达逐渐降低。3.Western blot结果显示,随着VX-680浓度的增加,凋亡分子Caspase3酶原蛋白水平逐渐降低,PARP分子出现明显的剪切带;粘附分子E-cadherin蛋白表达逐渐增强,CD44蛋白的表达逐渐减弱;基质金属蛋白酶MMP-2蛋白的表达逐渐减弱。4.Western blot结果显示:随着VX-680浓度的增加,p-ERK、p-AKT蛋白的表达降低。结论:1.Aurora激酶抑制剂VX-680可以抑制食管癌细胞的增殖、促进其凋亡及失巢凋亡、增强细胞间粘附能力、减弱细胞-外基质粘附能力及迁移能力,这种效应具有剂量效应关系,这种作用与Caspase3、PARP的活化程度及E-cadherin、CD44、MMP-2的表达有关。2.VX-680可能通过AKT和ERK通路而影响食管癌KYSE150细胞的增殖、粘附、迁移及失巢凋亡。
[Abstract]:Aim: to investigate the effects of Aurora kinase inhibitor VX-680 on the proliferation, apoptosis, adhesion, migration and apoptosis of esophageal carcinoma KYSE150 cells. Method 1: 1. MTT staining, intercellular adhesion, cell-extracellular matrix adhesion and scratch test were used to observe the effects of different concentrations of VX-680 on the proliferation, apoptosis, adhesion and migration of KYSE150 cells. The expression of adhesion molecule CD44 and matrix metalloproteinase MMP-2 was detected by Real-time PCR. Western blot technique was used to detect the expression of Caspase3, E-cadherin CD44, and matrix metalloproteinase MMP-2 protein in KYSE150 cells treated with different concentrations of VX-680. Western blot technique was used to detect the effect of different concentrations of VX-680 on the expression of AKT / p-AKT protein. The result is 1: 1. After treated with different concentrations of VX-680 on esophageal cancer cell line KYSE150, the cell proliferation rate decreased gradually with the increase of VX-680 concentration (P0.05). The rate of nuclear fragmentation gradually increased (P0.001); the cell adhesion test showed that the lumps formed by cell aggregation were larger and tighter (P0.001); the separation between cells became more difficult and the dispersion decreased (P0.001); and the cell-extracellular matrix adhesion assay showed that, The adhesion ability of cells to three kinds of extracellular matrix decreased gradually (P0.05), and the healing ability of cells decreased gradually (P0.001) .2.Real-time PCR results showed that the expression of CD44 and MMP-2 decreased gradually with the increase of VX-680 concentration. 3. Western blot results showed that: 1. With the increase of the concentration of VX-680, the level of Caspase3 proto protein decreased gradually, and the expression of E-cadherin protein gradually increased, and the expression of CD44 protein decreased gradually. The expression of matrix metalloproteinase (MMP-2) protein decreased gradually. 4. The results of Western blot showed that the expression of p-ERKN p-AKT protein decreased with the increase of VX-680 concentration. Conclusion: 1. VX-680 can inhibit the proliferation of esophageal cancer cells, promote apoptosis and apoptosis, enhance the adhesion between cells and extracellular matrix, and decrease the adhesion and migration ability of esophageal cancer cells. This effect has a dose-effect relationship. This effect is related to the activation of Caspase3PARP and the expression of E-cadherinon CD44-pMMP-2. 2. VX-680 may affect the proliferation, adhesion, migration and apoptosis of esophageal carcinoma KYSE150 cells through AKT and ERK pathways.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.1

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