PRPS1在儿童急性白血病中的表达及其作用机制的研究

发布时间：2018-01-26 08:40

本文关键词： 磷酸核糖焦磷酸合成酶1 急性白血病儿童危险因素 B细胞急性白血病磷酸核糖焦磷酸合成酶1 Bcl-2 增殖凋亡　出处：《重庆医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:探讨磷酸核糖焦磷酸合成酶1(PRPS1)基因在儿童急性白血病(AL)中的表达,分析其表达水平与AL临床指标的相关性,初步探讨PRPS1在AL中的临床意义。方法:采用实时荧光定量PCR和免疫组化(IH)的方法检测176例儿童AL(初诊126例,完全缓解50例)骨髓标本中PRPS1的表达水平,收集患儿病程中诊断结果,临床表现以及治疗反应等临床特征信息,分析其PRPS1表达水平与临床特征的相关性。以21例非恶性血液病患儿骨髓作为对照组患儿。结果:(1)在B-ALL组中,PRPS1初诊组表达水平显著高于对照组,同时高于完全缓解组(均P0.0001);在T-ALL及AML组中,仅初诊组与完全缓解组表达有显著差异(均P0.0001)。(2)在B-ALL组中,PRPS1表达水平随危险度升高而增加(P0.01);在T-ALL组中各危险度分组之间则无明显差异(P0.05);在AML组中,仅低危组与高危组差异具有统计学意义(P0.05)。(3)PRPS1高表达与B-ALL患儿的高WBC计数及MRD阳性相关(P=0.020,P=0.026)。(4)PRPS1与之前证实的复发相关基因NT5C2表达水平呈正相关(P0.05)。结论:PRPS1是儿童B-ALL预后不良相关危险因素,有望成为判断预后,指导个性化化疗的指标。目的:利用慢病毒上调PRPS1基因的表达,研究其对Sup-b15及Raji细胞株增殖、凋亡及细胞周期的调控作用及机制。方法:采用PRPS1过表达慢病毒感染Sup-b15及Raji细胞株构建实验组PRPS1过表达细胞;采用GFP过表达慢病毒感染Sup-b15及Raji细胞株作为对照组细胞;使用CCK-8法检测细胞增殖;流式细胞术检测细胞周期及凋亡;采用q RT-PCR、Western-blot方法检测PRPS1及增殖、凋亡相关基因c-myc,cyclin E1,Bcl-2,CDK2,and caspase-3基因和蛋白表达水平的变化。结果:(1)与对照组细胞相比,实验组细胞PRPS1 m RNA和蛋白表达水平均显著上升,P0.05;(2)CCK-8法检测细胞增殖结果显示,实验组Sup-b15及Raji细胞的增殖率均上升,与对照组相比,P0.001;(3)流式细胞学检测细胞凋亡和周期结果显示,与对照组相比,实验组Sup-b15及Raji细胞的凋亡率均下降,P0.01;细胞周期差异无统计学意义,P0.05。(4)与对照组相比,实验组Sup-b15及Raji细胞的Bcl-2,CDK2基因和蛋白表达水平均显著提高,P0.001;Caspase-3蛋白水平表达下调P0.01;C-myc及Cyclin E1未见明显变化。结论:PRPS1基因过表达对Sup-b15及Raji细胞有促进增殖、抑制凋亡的作用,其机制可能与上调Bcl-2,增强细胞的抗凋亡作用相关。
[Abstract]:Objective: to investigate the expression of ribonucleotide pyrophosphate synthase 1 (PRPS1) gene in children with acute leukemia (ALL) and to analyze the correlation between the expression level and the clinical parameters of AL. To explore the clinical significance of PRPS1 in AL. Methods: real time fluorescence quantitative PCR and immunohistochemistry were used to detect 176 children with ALS (126 cases first diagnosed). The expression level of PRPS1 was collected in 50 cases of complete remission), and the clinical characteristics, such as diagnosis, clinical manifestation and therapeutic response were collected. The correlation between the expression of PRPS1 and clinical features was analyzed. The bone marrow of 21 children with non-malignant hematologic disease was used as control group. Results: 1) in B-ALL group. The expression level of PRPS1 in the newly diagnosed group was significantly higher than that in the control group and in the complete remission group (all P 0.0001). In T-ALL and AML group, the expression of T-ALL was significantly different from that of complete remission group (all P0.0001. 2) in B-ALL group. The expression of PRPS1 increased with the increase of risk. In T-ALL group, there was no significant difference among the risk groups (P 0.05). In the AML group. The high expression of PRPS1 in low risk group and high risk group was significantly higher than that in high risk group. The high expression of PRPS1 was associated with high WBC count and MRD positive correlation with P0. 020 in children with B-ALL. There was a positive correlation between the expression level of PRPS1 and the previously confirmed recurrence related gene NT5C2 (P0.05). Conclusion 1: PRPS1 is a risk factor for poor prognosis of B-ALL in children. Objective: to study the effect of lentivirus on the proliferation of Sup-b15 and Raji cell lines by up-regulating the expression of PRPS1 gene. Methods: PRPS1 overexpression lentivirus was used to infect Sup-b15 and Raji cell lines to construct PRPS1 overexpression cells in experimental group. GFP overexpression lentivirus was used to infect Sup-b15 and Raji cell lines as control cells. Cell proliferation was detected by CCK-8 assay. Cell cycle and apoptosis were detected by flow cytometry. PRPS1, proliferation and apoptosis related gene c-myc cyclin E1Bcl-2 CDK2 were detected by Q RT-PCR Western-blot. Results compared with the control group, the expression of PRPS1 m RNA and protein in the experimental group was significantly higher than that in the control group. P0.05; The proliferation rate of Sup-b15 and Raji cells in the experimental group was higher than that in the control group (P 0.001). Flow cytometric analysis showed that the apoptotic rate of Sup-b15 and Raji cells in the experimental group was lower than that in the control group (P 0.01). There was no significant difference in cell cycle between the experimental group and the control group (P 0.05. 05. 4) the Bcl-2 of Sup-b15 and Raji cells in the experimental group was higher than that in the control group. The expression level of CDK2 gene and protein increased significantly (P 0.001). The expression of Caspase-3 protein down-regulated P0.01; C-myc and Cyclin E1 showed no significant changes. Conclusion the overexpression of the 1 / PRPS1 gene can promote the proliferation and inhibit the apoptosis of Sup-b15 and Raji cells. The mechanism may be related to the up-regulation of BCL-2 and the enhancement of the anti-apoptotic effect of BCL-2.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R733.71

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