本文關(guān)鍵詞：ATRA對(duì)K562細(xì)胞中H0XA5基因表達(dá)的影響及其與細(xì)胞周期、凋亡關(guān)系的研究　出處：《四川醫(yī)科大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： K562細(xì)胞 細(xì)胞凋亡 細(xì)胞周期 HOX5基因 全反式維甲酸

【摘要】：目的:本課題旨在通過(guò)全反式維甲酸(All-Trans Retinoic Acid,ATRA)對(duì)人髓系白血病細(xì)胞株K562細(xì)胞的干預(yù)來(lái)分析同源盒基因(Homeobox genes,HOX)A5的表達(dá)變化及其與凋亡率、細(xì)胞周期的關(guān)系,探討HOXA5在髓系白血病發(fā)生、發(fā)展過(guò)程中的作用。為臨床治療白血病提供實(shí)驗(yàn)依據(jù)。方法:1.采用腫瘤細(xì)胞體外培養(yǎng)技術(shù)培養(yǎng)人髓系白血病細(xì)胞K562細(xì)胞作為實(shí)驗(yàn)?zāi)Ｐ汀?.細(xì)胞增殖-毒性實(shí)驗(yàn)(Cell Counting Kit-8,CCK-8):測(cè)定用不同濃度的ATRA干預(yù)K562細(xì)胞24h、48h、72h、96h后,對(duì)K562細(xì)胞的增殖抑制情況,以確定ATRA干預(yù)K562細(xì)胞的最佳濃度值。3.實(shí)驗(yàn)分組:取對(duì)數(shù)生長(zhǎng)期細(xì)胞(約105/ml),將K562細(xì)胞隨機(jī)分為ATRA干預(yù)組(實(shí)驗(yàn)組)和對(duì)照組。實(shí)驗(yàn)組設(shè)立三個(gè)時(shí)間亞組,以10.0 umol/L的ATRA分別干預(yù)24h、48h和72h;對(duì)照組用培養(yǎng)基代替ATRA,其余同ATRA干預(yù)組。4.流式細(xì)胞術(shù)檢測(cè):測(cè)定10.0 umol/L ATRA分別干預(yù)K562細(xì)胞24h、48h、72h后,各組細(xì)胞的凋亡情況。5.流式細(xì)胞分析儀檢測(cè):檢測(cè)并分析10.0 umol/L ATRA干預(yù)K562細(xì)胞不同時(shí)間后,與相應(yīng)的對(duì)照組相比細(xì)胞周期分布的變化。6.實(shí)時(shí)熒光定量pcr(rt-pcr)技術(shù)檢測(cè):測(cè)定10.0umol/latra干預(yù)k562細(xì)胞24h、48h、72h后,細(xì)胞中hoxa5mrna的表達(dá)情況,并分析hoxa5mrna的表達(dá)與細(xì)胞周期、凋亡的關(guān)系。7.蛋白免疫印跡(westernblot)法檢測(cè):測(cè)定10.0umol/latra干預(yù)k562細(xì)胞24h、48h、72h后細(xì)胞中hoxa5蛋白的表達(dá)情況,并分析hoxa5蛋白表達(dá)與細(xì)胞周期、凋亡的關(guān)系。8.統(tǒng)計(jì)方法分析:將實(shí)驗(yàn)結(jié)果通過(guò)統(tǒng)計(jì)學(xué)spss17.0軟件處理,以p0.05為差異有統(tǒng)計(jì)學(xué)意義。兩變量間相關(guān)性分析采用spearman等級(jí)相關(guān)分析檢驗(yàn),a=0.05為顯著性檢驗(yàn)水準(zhǔn)。結(jié)果:1.cck-8結(jié)果顯示:atra可以抑制k562細(xì)胞的增殖,各實(shí)驗(yàn)組與對(duì)照組相比,od值的差異有統(tǒng)計(jì)學(xué)意義(p0.05)。atra干預(yù)濃度為7.5umol/l、10.0umol/l時(shí),各組細(xì)胞在不同時(shí)間點(diǎn)的生長(zhǎng)趨勢(shì)最佳。本實(shí)驗(yàn)最終選取10.0umol/l作為干預(yù)藥物濃度。2.流式細(xì)胞術(shù)檢測(cè)細(xì)胞凋亡率結(jié)果顯示:實(shí)驗(yàn)組細(xì)胞凋亡率明顯高于對(duì)照組(p0.05),且各實(shí)驗(yàn)組隨著藥物干預(yù)時(shí)間的延長(zhǎng),細(xì)胞凋亡率也逐漸增加(p0.05)。3.流式細(xì)胞術(shù)檢測(cè)細(xì)胞周期分布顯示:atra干預(yù)k562細(xì)胞24h、48h、72h后,g0/g1期比例增高,s期比例降低,細(xì)胞增殖周期被阻抑在g0/g1期,細(xì)胞增殖受到抑制。4.rt-pcr結(jié)果顯示:k562細(xì)胞中可以檢測(cè)到hoxa5mrna的表達(dá),實(shí)驗(yàn)組hoxa5mrna的表達(dá)量較對(duì)照組升高(p0.05),且隨著干預(yù)時(shí)間延長(zhǎng)各實(shí)驗(yàn)組間HOXA5 mRNA的表達(dá)量也逐漸增加(P0.05)。5.Western Blot結(jié)果顯示:HOXA5蛋白高表達(dá)于K562細(xì)胞中,ATRA干預(yù)組表達(dá)量較正常對(duì)照組高(P0.05),且隨著干預(yù)時(shí)間的延長(zhǎng)HOXA5蛋白的表達(dá)量逐漸升高(P0.05)。6.HOXA5的表達(dá)水平與細(xì)胞凋亡相關(guān)性顯示:K562細(xì)胞中HOXA5 mRNA及蛋白的表達(dá)量與細(xì)胞凋亡率的相關(guān)性經(jīng)Spearman等級(jí)相關(guān)分析顯示兩者呈正相關(guān)。7.HOXA5的表達(dá)水平與細(xì)胞周期相關(guān)性顯示:HOXA5 mRNA及蛋白的表達(dá)量與細(xì)胞周期G0/G1期增加百分比呈正相關(guān),與S期降低百分比呈負(fù)相關(guān)。結(jié)論:1.ATRA干預(yù)K562細(xì)胞后,細(xì)胞中HOXA5 mRNA及蛋白的表達(dá)量升高。2.ATRA可能通過(guò)上調(diào)HOXA5 mRNA及蛋白的表達(dá)抑制K562細(xì)胞增殖,促進(jìn)細(xì)胞凋亡。
[Abstract]:Purpose: the purpose of this topic by all trans retinoic acid (All-Trans Retinoic Acid, ATRA) on human myeloid leukemia cell line K562 cells to analyze the intervention of homeobox gene (Homeobox, genes, HOX) A5 expression and apoptosis rate, the relationship of cell cycle, explore the role of HOXA5 in myeloid leukemia, development process the role. Provide experimental basis for clinical treatment of leukemia. Methods: 1. the tumor cells cultured in vitro culture of human myeloid leukemia cells K562 cell proliferation as.2. cell toxicity experiment model (Cell Counting Kit-8, CCK-8): the determination of 24h, ATRA treatment of K562 cells with different concentrations of 48h, 72h, 96h. Inhibition of K562 cell proliferation, to determine the optimal concentration of ATRA intervention K562 cells.3. group: cells in the logarithmic growth phase (about 105/ml), the K562 cells were randomly divided into ATRA group (experimental group) and the control group. The experimental group set up three sub groups, with 10 umol/L ATRA respectively 24h 48h and 72h; intervention, control group with medium instead of ATRA, the rest of the ATRA intervention group.4. flow cytometry: Determination of 10 umol/L ATRA K562 cells were treated 24h, 48h, 72h, apoptosis of.5. cells flow cytometry: detection and analysis of 10 umol/L ATRA K562 cells in different time after intervention, compared with the control group, the corresponding change of.6. real-time fluorescence quantitative PCR cell cycle distribution (RT-PCR) detection technology: Determination of 10.0umol/latra intervention K562 cell 24h, 48h, 72h, the expression of hoxa5mrna in cells, and expression and analysis the cell cycle of hoxa5mrna,.7. protein and apoptosis (Westernblot) assay: Determination of 10.0umol/latra intervention K562 cell 24h, 48h, Hoxa5 protein expression in 72h cells, and the expression of Hoxa5 protein and cell analysis Cycle, analysis of the relationship between the.8. statistical method: the apoptosis of the experimental results by SPSS17.0 statistical software processing, with P0.05 were statistically analyzed using Spearman rank correlation analysis the correlation between the two variables, inspection level is a=0.05. Results: 1.cck-8 results showed that ATRA can inhibit the proliferation of K562 cells in each experimental group compared with the control group, significant difference in OD (P0.05).Atra on the concentration of 7.5umol/l, 10.0umol/l, the cells were the best in the growth trend of different time points. The final selection of 10.0umol /l as the intervention of drug concentration.2. flow cytometry to detect the apoptosis rate of results showed that the apoptosis rate of experimental group was significantly higher than that of the control group (P0.05), and the experimental group with prolonged drug intervention time, the apoptosis rate increased gradually (P0.05).3. flow cytometry to detect the cell cycle distribution. Show: ATRA 48h, 24h intervention K562 cells, after 72h, the proportion of g0/g1 phase increased and S phase decrease, cell cycle was inhibited in g0/g1 phase, cell proliferation was inhibited by.4.rt-pcr showed that K562 cells can detect the expression of hoxa5mrna, expression of hoxa5mrna in the experimental group was higher than the control group (P0.05). And with the expression of prolonged intervention among the experimental groups HOXA5 mRNA increased gradually (P0.05).5.Western Blot showed that the high expression of HOXA5 protein in K562 cells, the expression of ATRA in the intervention group were higher than normal control group (P0.05), and the expression of HOXA5 protein with prolonged intervention time increased gradually (P0.05) show the expression level and cell apoptosis in.6.HOXA5: correlation analysis of expression and apoptosis rate of mRNA HOXA5 and protein in K562 cells by Spearman rank correlation showed that the expression level of both Cheng Zhengxiang.7.HOXA5 and fine According to the cell cycle correlation: increased expression of cell cycle G0/G1 HOXA5 mRNA and protein was positively correlated with the percentage of negative correlation, and the percentage of S phase decreased. Conclusion: 1.ATRA K562 cells increased after the intervention, the mRNA and protein expression of HOXA5 cells in.2.ATRA could inhibit the proliferation of K562 cells by up regulating expression of HOXA5 protein and mRNA. To promote cell apoptosis.

【學(xué)位授予單位】：四川醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R733.7

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