Aurora激酶抑制劑VX-680對食管癌細胞增殖、粘附、遷移及失巢凋亡的影響
發(fā)布時間：2018-08-05 16:46
【摘要】：目的:觀察Aurora激酶抑制劑VX-680對食管癌KYSE150細胞增殖、凋亡、粘附、遷移及失巢凋亡的影響,并探討其分子機制。方法:1.采用MTT法、DAPI染色法、細胞間粘附實驗、細胞-細胞外基質粘附實驗、劃痕實驗觀察不同濃度(0μΜ、0.5μΜ、1μΜ)的VX-680對KYSE150細胞增殖、凋亡、失巢凋亡、粘附及遷移能力的影響;2.采用Real-time PCR技術檢測粘附分子CD44和基質金屬蛋白酶MMP-2的表達;3.采用Western blot技術檢測不同濃度的VX-680作用KYSE150細胞后凋亡分子Caspase3、PARP,粘附分子E-cadherin、CD44及影響遷移的基質金屬蛋白酶MMP-2分子蛋白表達情況;4.采用Western blot技術檢測不同濃度的VX-680作用后對ERK/p-ERK、AKT/p-AKT蛋白表達水平的影響。結果:1.不同濃度的VX-680作用于食管癌細胞株KYSE150后,發(fā)現(xiàn)隨著VX-680濃度的增加:MTT法實驗結果顯示細胞的增殖率逐漸降低(P0.05);DAPI染色法顯示,細胞發(fā)生核濃縮、核碎裂的比例逐漸增高(P0.001);細胞間粘附實驗顯示,細胞聚集形成的團塊更大,更緊密(P0.001);細胞間更難分離,離散程度逐漸減弱(P0.001);細胞-細胞外基質粘附實驗顯示,細胞與三種不同的外基質的粘附能力均逐漸減弱(P0.05);劃痕實驗結果顯示,細胞的愈合能力逐漸減弱(P0.001)。2.Real-time PCR結果顯示,隨著VX-680濃度的增加,CD44和MMP-2的表達逐漸降低。3.Western blot結果顯示,隨著VX-680濃度的增加,凋亡分子Caspase3酶原蛋白水平逐漸降低,PARP分子出現(xiàn)明顯的剪切帶;粘附分子E-cadherin蛋白表達逐漸增強,CD44蛋白的表達逐漸減弱;基質金屬蛋白酶MMP-2蛋白的表達逐漸減弱。4.Western blot結果顯示:隨著VX-680濃度的增加,p-ERK、p-AKT蛋白的表達降低。結論:1.Aurora激酶抑制劑VX-680可以抑制食管癌細胞的增殖、促進其凋亡及失巢凋亡、增強細胞間粘附能力、減弱細胞-外基質粘附能力及遷移能力,這種效應具有劑量效應關系,這種作用與Caspase3、PARP的活化程度及E-cadherin、CD44、MMP-2的表達有關。2.VX-680可能通過AKT和ERK通路而影響食管癌KYSE150細胞的增殖、粘附、遷移及失巢凋亡。
[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.
【學位授予單位】：山西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R735.1
本文編號：2166385
[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.
【學位授予單位】：山西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R735.1
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