發(fā)布時(shí)間：2018-08-05 16:46

【摘要】：目的:觀察Aurora激酶抑制劑VX-680對(duì)食管癌KYSE150細(xì)胞增殖、凋亡、粘附、遷移及失巢凋亡的影響,并探討其分子機(jī)制。方法:1.采用MTT法、DAPI染色法、細(xì)胞間粘附實(shí)驗(yàn)、細(xì)胞-細(xì)胞外基質(zhì)粘附實(shí)驗(yàn)、劃痕實(shí)驗(yàn)觀察不同濃度(0μΜ、0.5μΜ、1μΜ)的VX-680對(duì)KYSE150細(xì)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(shù)檢測(cè)粘附分子CD44和基質(zhì)金屬蛋白酶MMP-2的表達(dá);3.采用Western blot技術(shù)檢測(cè)不同濃度的VX-680作用KYSE150細(xì)胞后凋亡分子Caspase3、PARP,粘附分子E-cadherin、CD44及影響遷移的基質(zhì)金屬蛋白酶MMP-2分子蛋白表達(dá)情況;4.采用Western blot技術(shù)檢測(cè)不同濃度的VX-680作用后對(duì)ERK/p-ERK、AKT/p-AKT蛋白表達(dá)水平的影響。結(jié)果:1.不同濃度的VX-680作用于食管癌細(xì)胞株KYSE150后,發(fā)現(xiàn)隨著VX-680濃度的增加:MTT法實(shí)驗(yàn)結(jié)果顯示細(xì)胞的增殖率逐漸降低(P0.05);DAPI染色法顯示,細(xì)胞發(fā)生核濃縮、核碎裂的比例逐漸增高(P0.001);細(xì)胞間粘附實(shí)驗(yàn)顯示,細(xì)胞聚集形成的團(tuán)塊更大,更緊密(P0.001);細(xì)胞間更難分離,離散程度逐漸減弱(P0.001);細(xì)胞-細(xì)胞外基質(zhì)粘附實(shí)驗(yàn)顯示,細(xì)胞與三種不同的外基質(zhì)的粘附能力均逐漸減弱(P0.05);劃痕實(shí)驗(yàn)結(jié)果顯示,細(xì)胞的愈合能力逐漸減弱(P0.001)。2.Real-time PCR結(jié)果顯示,隨著VX-680濃度的增加,CD44和MMP-2的表達(dá)逐漸降低。3.Western blot結(jié)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(dá)逐漸增強(qiáng),CD44蛋白的表達(dá)逐漸減弱;基質(zhì)金屬蛋白酶MMP-2蛋白的表達(dá)逐漸減弱。4.Western blot結(jié)果顯示:隨著VX-680濃度的增加,p-ERK、p-AKT蛋白的表達(dá)降低。結(jié)論:1.Aurora激酶抑制劑VX-680可以抑制食管癌細(xì)胞的增殖、促進(jìn)其凋亡及失巢凋亡、增強(qiáng)細(xì)胞間粘附能力、減弱細(xì)胞-外基質(zhì)粘附能力及遷移能力,這種效應(yīng)具有劑量效應(yīng)關(guān)系,這種作用與Caspase3、PARP的活化程度及E-cadherin、CD44、MMP-2的表達(dá)有關(guān)。2.VX-680可能通過(guò)AKT和ERK通路而影響食管癌KYSE150細(xì)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(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R735.1

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