本文關(guān)鍵詞：AG490對(duì)紅白血病HEL細(xì)胞VEGF、HIF-1α表達(dá)的影響　出處：《承德醫(yī)學(xué)院》2016年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 骨髓增殖性腫瘤 AG490 血管新生 VEGF HIF-1α 遷移

【摘要】：目的:將不同濃度JAK2激酶抑制劑AG490作用于人紅白血病(human erythroleukemia,HEL)細(xì)胞,觀察其對(duì)HEL細(xì)胞增殖、凋亡,細(xì)胞遷移能力的影響及對(duì)VEGF、HIF-1α蛋白表達(dá)的變化,探討AG490能否通過抑制JAK2-STAT5信號(hào)通路抑制血管新生因子VEGF和HIF-1α表達(dá),為抗血管新生治療惡性血液病提供理論依據(jù)。方法:1.實(shí)驗(yàn)分組:空白對(duì)照組(未加用AG490組),實(shí)驗(yàn)組(加用不同濃度AG490組)。根據(jù)AG490不同終濃度,實(shí)驗(yàn)組又分為:20μmol/L AG490組,40μmol/L AG490組,60μmol/L AG490組,80μmol/L AG490組,100μmol/L AG490組。2.應(yīng)用Cell Counting Kit-8(CCK-8)法檢測(cè)AG490對(duì)HEL細(xì)胞活力的影響。3.應(yīng)用Hoechst熒光染色法觀察AG490對(duì)HEL細(xì)胞凋亡形態(tài)的變化。4.應(yīng)用流式細(xì)胞技術(shù)檢測(cè)AG490對(duì)HEL細(xì)胞凋亡的影響。5.Transwell小室實(shí)驗(yàn)檢測(cè)AG490對(duì)HEL細(xì)胞遷移的影響。6.應(yīng)用半定量-聚合酶鏈反應(yīng)(RT-PCR)檢測(cè)AG490對(duì)HEL細(xì)胞JAK2的m RNA表達(dá)水平的影響。7.應(yīng)用蛋白免疫印跡法(Western blot)檢測(cè)AG490對(duì)HEL細(xì)胞p-JAK2、VEGF及HIF-1α的蛋白表達(dá)水平的影響。8.統(tǒng)計(jì)學(xué)分析:數(shù)據(jù)以均數(shù)±標(biāo)準(zhǔn)差(`x±S)表示,通過SPSS19.00統(tǒng)計(jì)軟件分析。Transwell實(shí)驗(yàn)數(shù)據(jù)比較采用t檢驗(yàn),細(xì)胞活力、熒光染色、流式細(xì)胞術(shù)、RT-PCR和Western blot結(jié)果數(shù)據(jù)比較采用方差分析,組間數(shù)據(jù)比較選用q檢驗(yàn),兩變量的相關(guān)程度用Pearson直線相關(guān)分析法分析。P0.05表示差異有統(tǒng)計(jì)學(xué)意義。結(jié)果:1.CCK-8法檢測(cè)AG490對(duì)HEL細(xì)胞活力的影響CCK-8實(shí)驗(yàn)結(jié)果顯示:20μmol/L AG490組、40μmol/L AG490組、60μmol/L AG490組、80μmol/L AG490組、100μmol/L AG490組作用HEL細(xì)胞后,在不同時(shí)間各組細(xì)胞活力如下:24h時(shí)分別為:(97.2±0.6)%、(82.4±0.5)%、(70.5±0.5)%、(59.2±0.2)%、(41.9±0.3)%;48h時(shí)分別為:(88.3±0.5)%、(75.1±0.4)%、(48.8±0.6)%、(21.2±0.2)%、(10.2±0.1)%;72h時(shí)分別為:(69.9±0.4)%、(31.4±0.2)%、(19.6±0.3)%、(5.4±0.1)%、(1.5±0.1)%。隨著孵育時(shí)間的延長(zhǎng),實(shí)驗(yàn)組HEL細(xì)胞活力呈現(xiàn)明顯降低趨勢(shì),與空白對(duì)照組相比,差異有統(tǒng)計(jì)學(xué)意義(P0.05);同一時(shí)間不同實(shí)驗(yàn)組HEL細(xì)胞活力明顯不同,且隨著AG490藥物濃度不斷增大,HEL細(xì)胞活力明顯降低。2.Hoechst33342熒光染色法觀察AG490作用于HEL細(xì)胞后細(xì)胞形態(tài)的變化熒光染色結(jié)果顯示:處理HEL細(xì)胞48h后,實(shí)驗(yàn)組HEL細(xì)胞出現(xiàn)凋亡,細(xì)胞核由于濃集而呈現(xiàn)亮藍(lán)色,核固縮或核變形�？瞻讓�(duì)照組HEL細(xì)胞Hoechst著色為淡藍(lán)色,細(xì)胞核內(nèi)DNA分布均勻,核呈圓形或者卵圓形,無(wú)固縮,無(wú)變形。空白對(duì)照組、20μmol/L AG490組、40μmol/L AG490組、60μmol/L AG490組、80μmol/L AG490組、100μmol/L AG490組亮藍(lán)色細(xì)胞數(shù)分別為:10.2±0.8、15.3±0.9、21.6±1.2、34.8±1.6、49.2±1.9、63.3±2.4,實(shí)驗(yàn)組與空白對(duì)照組相比,亮藍(lán)色細(xì)胞明顯增多,并且隨著藥物濃度的增加,亮藍(lán)色細(xì)胞數(shù)量明顯增加,且有顯著性差異(P0.05)。3.流式細(xì)胞術(shù)檢測(cè)AG490對(duì)HEL細(xì)胞凋亡的影響流式細(xì)胞術(shù)檢測(cè)結(jié)果顯示:處理HEL細(xì)胞48h后,空白對(duì)照組、20μmol/L AG490組、40μmol/L AG490組、60μmol/L AG490組、80μmol/L AG490組、100μmol/L AG490組HEL細(xì)胞凋亡率分別為:(2.44±0.16)%、(5.34±0.31)%、(8.71±0.51)%、(10.22±0.83)%、(16.63±1.22)%、(23.33±1.40)%。與空白對(duì)照組相比,實(shí)驗(yàn)組細(xì)胞出現(xiàn)明顯凋亡,并且隨著AG490藥物濃度的增加,凋亡細(xì)胞比率逐漸上升(P0.05)。4.Transwell小室實(shí)驗(yàn)檢測(cè)AG490對(duì)HEL細(xì)胞遷移的影響結(jié)果顯示,20μmol/L AG490作用細(xì)胞24h后漏入下室的細(xì)胞數(shù)(51.6±2.9)明顯少于空白對(duì)照組(71.6±4.5),具有統(tǒng)計(jì)學(xué)差異(P0.05)。5.RT-PCR方法檢測(cè)AG490對(duì)HEL細(xì)胞JAK2 m RNA表達(dá)水平的影響RT-PCR結(jié)果顯示:處理細(xì)胞48h后,空白對(duì)照組、20μmol/L AG490組、40μmol/L AG490組、60μmol/L AG490組、80μmol/L AG490組、100μmol/L AG490組HEL細(xì)胞JAK2 m RNA相對(duì)表達(dá)水平分別為:0.98±0.07、0.93±0.06、0.65±0.03、0.52±0.02、0.32±0.02、0.12±0.01。實(shí)驗(yàn)組JAK2的m RNA表達(dá)較空白對(duì)照組明顯減低,并隨著藥物濃度的增加而逐漸減低,較空白對(duì)照組有統(tǒng)計(jì)學(xué)意義(P0.05)。6.Western blot法檢測(cè)AG490對(duì)HEL細(xì)胞p-JAK2、VEGF、HIF-1α的蛋白表達(dá)水平的影響Western blot結(jié)果顯示:孵育細(xì)胞48小時(shí)后,空白對(duì)照組、20μmol/L AG490組、40μmol/L AG490組、60μmol/L AG490組、80μmol/L AG490組、100μmol/L AG490組p-JAK2蛋白條帶相對(duì)灰度分別為: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蛋白條帶相對(duì)灰度分別為: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蛋白條帶相對(duì)灰度分別為: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蛋白表達(dá)水平在空白對(duì)照組和各實(shí)驗(yàn)組中無(wú)明顯差異。實(shí)驗(yàn)組HEL細(xì)胞p-JAK2蛋白水平逐漸減低的,均明顯低于空白對(duì)照組。VEGF和HIF-1α的蛋白水平隨著藥物濃度的增加逐漸下降,較空白對(duì)照組有統(tǒng)計(jì)學(xué)意義(P0.05)。相關(guān)性分析結(jié)果:VEGF與p-JAK2相關(guān)系數(shù)r=0.991,二者呈正相關(guān)(P0.05);HIF-1α與p-JAK2相關(guān)系數(shù)r=0.993,亦呈正相關(guān)性(P0.05)。結(jié)論:1 HEL細(xì)胞存在JAK2V617F突變及JAK2信號(hào)通路活化。2 JAK2抑制劑AG490能夠抑制HEL細(xì)胞活力,且具有時(shí)間及劑量依賴性。3 JAK2抑制劑AG490能誘導(dǎo)HEL細(xì)胞凋亡。4 AG490能夠抑制HEL細(xì)胞的遷移能力。5 AG490可通過抑制JAK2信號(hào)通路,明顯抑制p-JAK2、VEGF和HIF-1α的表達(dá),并進(jìn)一步參與抑制血管新生作用。
[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
【學(xué)位授予單位】：承德醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R733.7

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1 徐倩;AG490對(duì)紅白血病HEL細(xì)胞VEGF、HIF-1α表達(dá)的影響[D];承德醫(yī)學(xué)院;2016年

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