AG490对红白血病HEL细胞VEGF、HIF-1α表达的影响

发布时间：2017-12-28 14:05

本文关键词：AG490对红白血病HEL细胞VEGF、HIF-1α表达的影响　出处：《承德医学院》2016年硕士论文　论文类型：学位论文

【摘要】：目的:将不同浓度JAK2激酶抑制剂AG490作用于人红白血病(human erythroleukemia,HEL)细胞,观察其对HEL细胞增殖、凋亡,细胞迁移能力的影响及对VEGF、HIF-1α蛋白表达的变化,探讨AG490能否通过抑制JAK2-STAT5信号通路抑制血管新生因子VEGF和HIF-1α表达,为抗血管新生治疗恶性血液病提供理论依据。方法:1.实验分组:空白对照组(未加用AG490组),实验组(加用不同浓度AG490组)。根据AG490不同终浓度,实验组又分为:20μmol/L AG490组,40μmol/L AG490组,60μmol/L AG490组,80μmol/L AG490组,100μmol/L AG490组。2.应用Cell Counting Kit-8(CCK-8)法检测AG490对HEL细胞活力的影响。3.应用Hoechst荧光染色法观察AG490对HEL细胞凋亡形态的变化。4.应用流式细胞技术检测AG490对HEL细胞凋亡的影响。5.Transwell小室实验检测AG490对HEL细胞迁移的影响。6.应用半定量-聚合酶链反应(RT-PCR)检测AG490对HEL细胞JAK2的m RNA表达水平的影响。7.应用蛋白免疫印迹法(Western blot)检测AG490对HEL细胞p-JAK2、VEGF及HIF-1α的蛋白表达水平的影响。8.统计学分析:数据以均数±标准差(`x±S)表示,通过SPSS19.00统计软件分析。Transwell实验数据比较采用t检验,细胞活力、荧光染色、流式细胞术、RT-PCR和Western blot结果数据比较采用方差分析,组间数据比较选用q检验,两变量的相关程度用Pearson直线相关分析法分析。P0.05表示差异有统计学意义。结果:1.CCK-8法检测AG490对HEL细胞活力的影响CCK-8实验结果显示:20μmol/L AG490组、40μmol/L AG490组、60μmol/L AG490组、80μmol/L AG490组、100μmol/L AG490组作用HEL细胞后,在不同时间各组细胞活力如下:24h时分别为:(97.2±0.6)%、(82.4±0.5)%、(70.5±0.5)%、(59.2±0.2)%、(41.9±0.3)%;48h时分别为:(88.3±0.5)%、(75.1±0.4)%、(48.8±0.6)%、(21.2±0.2)%、(10.2±0.1)%;72h时分别为:(69.9±0.4)%、(31.4±0.2)%、(19.6±0.3)%、(5.4±0.1)%、(1.5±0.1)%。随着孵育时间的延长,实验组HEL细胞活力呈现明显降低趋势,与空白对照组相比,差异有统计学意义(P0.05);同一时间不同实验组HEL细胞活力明显不同,且随着AG490药物浓度不断增大,HEL细胞活力明显降低。2.Hoechst33342荧光染色法观察AG490作用于HEL细胞后细胞形态的变化荧光染色结果显示:处理HEL细胞48h后,实验组HEL细胞出现凋亡,细胞核由于浓集而呈现亮蓝色,核固缩或核变形。空白对照组HEL细胞Hoechst着色为淡蓝色,细胞核内DNA分布均匀,核呈圆形或者卵圆形,无固缩,无变形。空白对照组、20μmol/L AG490组、40μmol/L AG490组、60μmol/L AG490组、80μmol/L AG490组、100μmol/L AG490组亮蓝色细胞数分别为:10.2±0.8、15.3±0.9、21.6±1.2、34.8±1.6、49.2±1.9、63.3±2.4,实验组与空白对照组相比,亮蓝色细胞明显增多,并且随着药物浓度的增加,亮蓝色细胞数量明显增加,且有显著性差异(P0.05)。3.流式细胞术检测AG490对HEL细胞凋亡的影响流式细胞术检测结果显示:处理HEL细胞48h后,空白对照组、20μmol/L AG490组、40μmol/L AG490组、60μmol/L AG490组、80μmol/L AG490组、100μmol/L AG490组HEL细胞凋亡率分别为:(2.44±0.16)%、(5.34±0.31)%、(8.71±0.51)%、(10.22±0.83)%、(16.63±1.22)%、(23.33±1.40)%。与空白对照组相比,实验组细胞出现明显凋亡,并且随着AG490药物浓度的增加,凋亡细胞比率逐渐上升(P0.05)。4.Transwell小室实验检测AG490对HEL细胞迁移的影响结果显示,20μmol/L AG490作用细胞24h后漏入下室的细胞数(51.6±2.9)明显少于空白对照组(71.6±4.5),具有统计学差异(P0.05)。5.RT-PCR方法检测AG490对HEL细胞JAK2 m RNA表达水平的影响RT-PCR结果显示:处理细胞48h后,空白对照组、20μmol/L AG490组、40μmol/L AG490组、60μmol/L AG490组、80μmol/L AG490组、100μmol/L AG490组HEL细胞JAK2 m RNA相对表达水平分别为:0.98±0.07、0.93±0.06、0.65±0.03、0.52±0.02、0.32±0.02、0.12±0.01。实验组JAK2的m RNA表达较空白对照组明显减低,并随着药物浓度的增加而逐渐减低,较空白对照组有统计学意义(P0.05)。6.Western blot法检测AG490对HEL细胞p-JAK2、VEGF、HIF-1α的蛋白表达水平的影响Western blot结果显示:孵育细胞48小时后,空白对照组、20μmol/L AG490组、40μmol/L AG490组、60μmol/L AG490组、80μmol/L AG490组、100μmol/L AG490组p-JAK2蛋白条带相对灰度分别为:1.228±0.090,1.185±0.081,1.020±0.065,0.915±0.063,0.693±0.035,0.286±0.012。VEGF蛋白条带相对灰度分别为:1.029±0.051,0.896±0.043,0.743±0.021,0.598±0.019,0.465±0.012,0.214±0.005;HIF-1α蛋白条带相对灰度分别为:0.977±0.048,0.893±0.037,0.702±0.052,0.658±0.045,0.427±0.021,0.259±0.010,β-actin蛋白表达水平在空白对照组和各实验组中无明显差异。实验组HEL细胞p-JAK2蛋白水平逐渐减低的,均明显低于空白对照组。VEGF和HIF-1α的蛋白水平随着药物浓度的增加逐渐下降,较空白对照组有统计学意义(P0.05)。相关性分析结果:VEGF与p-JAK2相关系数r=0.991,二者呈正相关(P0.05);HIF-1α与p-JAK2相关系数r=0.993,亦呈正相关性(P0.05)。结论:1 HEL细胞存在JAK2V617F突变及JAK2信号通路活化。2 JAK2抑制剂AG490能够抑制HEL细胞活力,且具有时间及剂量依赖性。3 JAK2抑制剂AG490能诱导HEL细胞凋亡。4 AG490能够抑制HEL细胞的迁移能力。5 AG490可通过抑制JAK2信号通路,明显抑制p-JAK2、VEGF和HIF-1α的表达,并进一步参与抑制血管新生作用。
[Abstract]:Objective: different concentration of JAK2 kinase inhibitor AG490 in human erythroleukemia (human erythroleukemia HEL) cells, to observe the proliferation and apoptosis of HEL cells, influence cell migration and changes of expression of HIF-1 protein, VEGF, to investigate whether AG490 inhibit angiogenesis factor VEGF and HIF-1 expression inhibition of JAK2-STAT5 signaling and to provide a theoretical basis for anti angiogenesis treatment of malignant hematological diseases. Methods: 1. groups of experimental groups: blank control group (group without AG490), experimental group (group with different concentration of AG490). According to the different final concentrations of AG490, the experimental group was divided into 20 mu mol/L AG490 group, 40 mol/L AG490 group, 60 mu mol/L AG490 group, 80 mu mol/L AG490 group, 100 mu mol/L AG490 group. 2. Cell Counting Kit-8 (CCK-8) was used to detect the effect of AG490 on the activity of HEL cells. 3. Hoechst fluorescence staining was used to observe the changes in the apoptosis morphology of AG490 cells in HEL cells. 4. the effect of AG490 on the apoptosis of HEL cells was detected by flow cytometry. The effect of AG490 on the migration of HEL cells was detected by 5.Transwell laboratory test. 6. the effect of AG490 on the level of M RNA expression of JAK2 in HEL cells was detected by semi quantitative polymerase chain reaction (RT-PCR). 7. the effect of AG490 on the protein expression level of p-JAK2, VEGF and HIF-1 alpha in HEL cells was detected by Western blot. 8. statistical analysis: the data were expressed with mean standard deviation (`x + S), and analyzed by SPSS19.00 statistical software. Transwell test data were compared with t test. Cell viability, fluorescence staining, flow cytometry, RT-PCR and Western blot data were compared with ANOVA. The Q test was used to compare the data between two groups. The correlation between the two variables was analyzed by Pearson linear correlation analysis. P0.05 indicated that the difference was statistically significant. Results: the experimental results of AG490 1.CCK-8 detection method and effect on HEL cell viability of CCK-8 display: 20 mol/L AG490 40 mol/L group, AG490 group, AG490 group, 60 mol/L 80 mol/L AG490 100 mol/L group, AG490 group HEL cells, cell viability in different time are as follows: 24h respectively. (97.2 + 0.6)% and (82.4 + 0.5)% and (70.5 + 0.5)% and (59.2 + 0.2)% and (41.9 + 0.3)%; 48h respectively: (88.3 + 0.5)% and (75.1 + 0.4)% and (48.8 + 0.6)%, (21.2. 0.2)% and (10.2 + 0.1)%; 72h respectively: (69.9 + 0.4)% and (31.4 + 0.2)% and (19.6 + 0.3)% and (5.4 + 0.1)% and (1.5 + 0.1)%. With prolonging of incubation time, experimental group HEL cells activity showed significantly decreased compared with the control group, the difference was statistically significant (P0.05); the same time the different experimental group HEL cell viability was significantly different, and with the AG490 concentration increasing, HEL cell viability decreased significantly. To observe the effect of AG490 on the fluorescence change of cell morphology of HEL cells after 2.Hoechst33342 fluorescent staining staining results showed: HEL cells treated with 48h, experimental group HEL cells apoptosis, and showed a bright blue concentration due to nuclear, nuclear pyknosis or nuclear deformation. The Hoechst coloring of HEL cells in the blank control group was light blue, the distribution of DNA in the nucleus was uniform, and the nucleus was round or oval, and there was no contraction and no deformation. The blank control group, 20 mol/L group, AG490 group, AG490 40 mol/L 60 mol/L AG490 80 mol/L group, AG490 group, 100 mol/L group AG490 bright blue cell number was 10.2 + 0.8, 15.3 + 0.9, 21.6 + 1.2, 34.8 + 1.6, 49.2 + 1.9, 63.3 + 2.4. The experimental group and the control group, the bright blue cells increased obviously, and with the increase of drug concentration, bright blue cells increased significantly, and there is a significant difference (P0.05). Effect of AG490 3. flow cytometry detection on the apoptosis of HEL cells by flow cytometry showed that HEL cells treated with 48h, control group, AG490 group, 20 mol/L 40 mol/L AG490 60 mol/L group, AG490 group, AG490 group, 80 mol/L 100 mol/L AG490 group, the apoptosis rate of HEL cells respectively: (2.44 + 0.16)% and (5.34 + 0.31)% and (8.71 + 0.51)% and (10.22 + 0.83)% and (16.63 + 1.22)% and (23.33 + 1.40)%. Compared with the blank control group, the cells in the experimental group showed obvious apoptosis, and the percentage of apoptotic cells increased gradually with the increase of AG490 drug concentration (P0.05). The effect of AG490 on migration of HEL cells was detected by 4.Transwell cell experiment. The results showed that the number of cells leaking into the inferior chamber after 20 24h mol/L treatment of 24h cells (51.6 + 2.9) was significantly less than that in the blank control group (71.6 + 4.5), and the difference was statistically significant (P0.05). 5.RT-PCR method for detection of AG490 on HEL cells of JAK2 m RNA on the expression of RT-PCR showed that cells treated with 48h, control group, AG490 group, 20 mol/L 40 mol/L AG490 60 mol/L group, AG490 group, AG490 group, 80 mol/L 100 mol/L AG490 JAK2 m RNA HEL cells on the expression of the level was 0.98 + 0.07, 0.93 + 0.06, 0.65 + 0.03, 0.52 + 0.02, 0.32 + 0.02, 0.12 + 0.01. The m RNA expression of JAK2 in experimental group was significantly lower than that in blank control group, and decreased gradually with the increase of drug concentration, which was statistically significant compared with that in blank control group (P0.05). Western blot AG490 6.Western blot method for detection of p-JAK2, VEGF, HEL cells HIF-1 alpha protein expression levels showed that the cells were incubated for 48 hours, the blank control group, 20 mol/L group, AG490 group, AG490 40 mol/L 60 mol/L AG490 80 mol/L group, AG490 group, 100 mol/L AG490 group of p-JAK2 protein bands with relative gray respectively: 1.228 + 0.090,1.185 + 0.081,1.020 + 0.065,0.915 + 0.063,0.693 + 0.035,0.286 + 0.012. VEGF protein bands with relative gray respectively: 1.029 + 0.051,0.896 + 0.043,0.743 + 0.021,0.598 + 0.019,0.465 + 0.012,0.214 + 0.005; HIF-1 protein bands with relative gray respectively: 0.977 + 0.048,0.893 + 0.037,0.702 + 0.052,0.658 + 0.045,0.427 + 0.021,0.259 + 0.010, beta -actin protein expression in the control group and the experimental group had no obvious the difference. The level of p-JAK2 protein in HEL cells decreased gradually in the experimental group, which was significantly lower than that in the blank control group. VEGF and HIF-1 alpha
【学位授予单位】：承德医学院
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R733.7

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