ATRA对K562细胞中H0XA5基因表达的影响及其与细胞周期、凋亡关系的研究

发布时间：2018-01-12 08:14

本文关键词：ATRA对K562细胞中H0XA5基因表达的影响及其与细胞周期、凋亡关系的研究　出处：《四川医科大学》2015年硕士论文　论文类型：学位论文

【摘要】：目的:本课题旨在通过全反式维甲酸(All-Trans Retinoic Acid,ATRA)对人髓系白血病细胞株K562细胞的干预来分析同源盒基因(Homeobox genes,HOX)A5的表达变化及其与凋亡率、细胞周期的关系,探讨HOXA5在髓系白血病发生、发展过程中的作用。为临床治疗白血病提供实验依据。方法:1.采用肿瘤细胞体外培养技术培养人髓系白血病细胞K562细胞作为实验模型。2.细胞增殖-毒性实验(Cell Counting Kit-8,CCK-8):测定用不同浓度的ATRA干预K562细胞24h、48h、72h、96h后,对K562细胞的增殖抑制情况,以确定ATRA干预K562细胞的最佳浓度值。3.实验分组:取对数生长期细胞(约105/ml),将K562细胞随机分为ATRA干预组(实验组)和对照组。实验组设立三个时间亚组,以10.0 umol/L的ATRA分别干预24h、48h和72h;对照组用培养基代替ATRA,其余同ATRA干预组。4.流式细胞术检测:测定10.0 umol/L ATRA分别干预K562细胞24h、48h、72h后,各组细胞的凋亡情况。5.流式细胞分析仪检测:检测并分析10.0 umol/L ATRA干预K562细胞不同时间后,与相应的对照组相比细胞周期分布的变化。6.实时荧光定量pcr(rt-pcr)技术检测:测定10.0umol/latra干预k562细胞24h、48h、72h后,细胞中hoxa5mrna的表达情况,并分析hoxa5mrna的表达与细胞周期、凋亡的关系。7.蛋白免疫印迹(westernblot)法检测:测定10.0umol/latra干预k562细胞24h、48h、72h后细胞中hoxa5蛋白的表达情况,并分析hoxa5蛋白表达与细胞周期、凋亡的关系。8.统计方法分析:将实验结果通过统计学spss17.0软件处理,以p0.05为差异有统计学意义。两变量间相关性分析采用spearman等级相关分析检验,a=0.05为显著性检验水准。结果:1.cck-8结果显示:atra可以抑制k562细胞的增殖,各实验组与对照组相比,od值的差异有统计学意义(p0.05)。atra干预浓度为7.5umol/l、10.0umol/l时,各组细胞在不同时间点的生长趋势最佳。本实验最终选取10.0umol/l作为干预药物浓度。2.流式细胞术检测细胞凋亡率结果显示:实验组细胞凋亡率明显高于对照组(p0.05),且各实验组随着药物干预时间的延长,细胞凋亡率也逐渐增加(p0.05)。3.流式细胞术检测细胞周期分布显示:atra干预k562细胞24h、48h、72h后,g0/g1期比例增高,s期比例降低,细胞增殖周期被阻抑在g0/g1期,细胞增殖受到抑制。4.rt-pcr结果显示:k562细胞中可以检测到hoxa5mrna的表达,实验组hoxa5mrna的表达量较对照组升高(p0.05),且随着干预时间延长各实验组间HOXA5 mRNA的表达量也逐渐增加(P0.05)。5.Western Blot结果显示:HOXA5蛋白高表达于K562细胞中,ATRA干预组表达量较正常对照组高(P0.05),且随着干预时间的延长HOXA5蛋白的表达量逐渐升高(P0.05)。6.HOXA5的表达水平与细胞凋亡相关性显示:K562细胞中HOXA5 mRNA及蛋白的表达量与细胞凋亡率的相关性经Spearman等级相关分析显示两者呈正相关。7.HOXA5的表达水平与细胞周期相关性显示:HOXA5 mRNA及蛋白的表达量与细胞周期G0/G1期增加百分比呈正相关,与S期降低百分比呈负相关。结论:1.ATRA干预K562细胞后,细胞中HOXA5 mRNA及蛋白的表达量升高。2.ATRA可能通过上调HOXA5 mRNA及蛋白的表达抑制K562细胞增殖,促进细胞凋亡。
[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.

【学位授予单位】：四川医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R733.7

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