【作者】 张冰；

【导师】 鞠秀丽；

【作者基本信息】 山东大学 ， 儿科学， 2018， 博士

【摘要】 研究背景:前B细胞型急性淋巴细胞白血病(Precursor B-cell acute lymphoblastic leukemia,pre-B-ALL)是儿童常见的恶性疾病,虽然目前长期无病生存率大幅提高,但仍有20%-30%的患儿最终死亡,究其原因在于化疗过程中的不缓解以及复发。近年来,对于pre-B-ALL发生和发展的核酸、蛋白水平的调控机制研究已有大量文献报道,但是在糖基化异常方面报道较少,尤其是关于O-GlcNAc糖基化尚未见报道。O-GlcNAc糖基化是一种重要的蛋白质翻译后修饰,是由Gerald W.Hart首次发现。近年来,已经发现蛋白存在上百种翻译后修饰,其中糖基化修饰,尤其是O-GlcNAc糖基化修饰尤为引人注目。O-GlcNAc糖基化是一个动态可逆的过程,由乙酰氨基葡萄糖转移酶(β-N-acetylglucosaminyltransferase,OGT)负责糖基化,N-乙酰氨基葡萄糖苷酶(β-Nacetylglucosaminidase,OGA)负责去糖基化。在生物体内,O-GlcNAc糖基化控制基本的生命过程,例如蛋白质的合成、染色质的结构、DNA脱甲基化以及生物昼夜规律。一些信号蛋白如AKT、转录因子如c-Myc、p53和NF-κB都存在O-GlcNAc糖基化修饰。近年来,大量文献都证明了 O-GlcNAc糖基化蛋白在多种癌变中发挥重要作用,如乳腺癌、前列腺癌、膀胱癌等,但并没有在pre-B-ALL进行相关研究。本研究选用临床初发的pre-B-ALL患儿骨髓标本及pre-B-ALL细胞系Nalm-6细胞作为主要研究对象,从糖生物学角度分析O-GlcNAc糖基化及相关酶OGT、OGA在B-ALL发生发展的作用,并从细胞糖酵解代谢的角度探讨其作用的机制。研究目的:1.研究O-GlcNAc糖基化及相关催化酶在pre-B-ALL儿童白血病细胞中的表达水平及对细胞生物学行为的影响。2.探索异常O-GlcNAc糖基化通过PI3K/Akt/c-Myc通路在Nalm-6细胞糖酵解过程的调控作用。3.探讨联合应用糖酵解抑制剂2-DG及靶向抑制OGT对Nalm-6杀伤效果的影响。研究方法:1.研究O-GlcNAc糖基化及相关催化酶在pre-B-ALL儿童白血病细胞中的表达水平及对细胞生物学行为的影响1.1.骨髓标本采集自从2015年9月至2016年9月期间就诊的pre-B-ALL初诊患儿。标本采集使用肝素抗凝试管,采集后立即应用淋巴细胞分离液依据密度梯度离心法分离骨髓单个核细胞(Bone marrow mononuclear cells,BM-MNCs),用免疫磁珠进行阳性分选出CD19+的B淋巴细胞,流式细胞术检测分选纯度。1.2.采用Western-Blotting法检测pre-B-ALL患儿组和健康儿童组整体O-GlcNAc糖基化水平及OGT、OGA的蛋白表达水平。1.3以急性前B淋巴细胞白血病细胞系Nalm-6细胞为研究对象,分别应用小干扰RNA技术和OGT特异性抑制剂Alloxan处理Nalm-6细胞进行对生物学行为影响的检测。小干扰RNA法:合成针对OGT基因的siRNA(OGT-siRNA)和阴性对照的NC-siRNA,用 Lipofectamine2000 将 OGT-siRNA 和 NC-siRNA 转染至 Nalm-6细胞中,作用48h后,应用RT-PCR和Western-Bloting技术检测OGT的表达以及O-GlcNAc糖基化水平的变化以检测干扰效果。抑制剂法:以浓度为0、5、1Ommol/L的Alloxan作用于Nalm-6细胞24h后,Western-Bloting技术检测O-GlcNAc糖基化水平、OGT表达水平以验证Alloxan对OGT的抑制作用;验证抑制效果后,后续以以浓度为10mmol/L的Alloxan作用于Nalm-6细胞后以检测对生物学行为的影响。1.4 细胞增殖:采用 CCK-8 检测抑制 OGT(OGT-siRNA or Alloxan)24h、48h、72h、96h的波长为450nm处的吸光度值,并绘制相应的增殖曲线。1.5细胞凋亡:以AnnexinV-PI双染法标记,用流式细胞术检测OGT-siRNA作用48h,Alloxan作用24h时的细胞凋亡比例。2.探索异常O-GlcNAc糖基化通过PI3K/Akt/c-Myc通路在Nalm-6细胞糖酵解过程的调控作用2.1验证c-Myc受PI3K/Akt通路调控:使用不同浓度(0、1、5、10μmol/L)的LY294002(PI3K/Akt 通路抑制剂)处理 Nalm-6 细胞 48h 后,以 Western-Blotting法检测 Akt、磷酸化 Akt(AktSer473)、c-Myc、磷酸化 c-Myc(c-MycThr58)的蛋白表达情况。2.2比较低血清乳酸脱氢酶(L-LDH)的ALL患儿组CD19+细胞及血清乳酸脱氢酶(H-LDH)患儿组的PI3K/Akt/c-Myc的激活情况:以400u/L为分界点区别L-LDH组与H-LDH组,检测方法同2.1。2.3糖酵解活跃度检测:以不同浓度(0、1、5、10μmol/L)的LY294002处理Nalm-6细胞48h,用seahorse XFe24细胞能量分析仪检测细胞外酸化率(the extracellular acidification rate,ECAR),并统计分析 ECAR 基础值(baseline)、糖酵解能力(glycolytic capacity)、糖酵解储备(glycolytic reserve)的变化。2.4分析L-LDH和H-LDH白血病患儿的O-GlcNAc糖基化水平和OGT、OGA的表达。2.5糖酵解活跃程度检测:将Nalm-6细胞分为三组:实验组,即转染OGT-siRNA组,称为OGT-siRNA组;阴性对照组,即转染negative control siRNA组,称为NC-siRNA组;空白对照组,即只加入等量转染试剂组,称为control组。检测以上三组的ECAR,具体同2.3。2.6糖酵解催化过程相关分子检测:用实时荧光定量PCR检测以上3组HK2、Glut1、HIF-1α、LDHA、PFK1的mRNA表达水平,以β-actin为内参进行统计学分析。2.7检测PI3K/Akt/c-Myc通路激活情况:检测以上三组的该通路关键分子的磷酸化水平,检测方法同4.1。2.8检测IGF-1对OGT-siRNA所导致的糖酵解抑制作用的影响:OGT-siRNA+IGF-1 组:同时将 OGT-siRNA 和 IGF-1 作用于 Nalm-6 细胞 48h,OGT-siRNA组:只应用OGT-siRNA作用Nalm-6细胞48h,control组只加转染试剂,糖酵解检测同4.3。3.探讨联合应用糖酵解抑制剂2-DG及靶向抑制OGT对Nalm-6杀伤效果的影响3.1检测2-DG对Nalm-6细胞O-GlcNAc糖基化及OGT的影响:以Western-Blotting 法检测不同浓度(0、2.5、5mmol/L)的 2-DG 对 O-GlcNAc 糖基化及OGT蛋白表达的影响。3.2检测联合应用2-DG及靶向抑制OGT对Nalm-6细胞增殖的影响:该部分分为3 组:2-DG 组为加 2-DG(2.5 mmol/L)处理组;2-DG+NC 组为 2-DG(2.5 mmol/L)及 NC-siRNA 处理组;2-DG+OGT-siRNA 组为联合使用用 2-DG(2.5 mmol/L)及OGT-siRNA处理组;检测增殖方法同1.4。3.3检测联合应用2-DG及靶向抑制OGT对细胞凋亡的影响:分组同3.2,以Annexin V-FITC染色结合流式细胞术检测细胞凋亡情况。结果:1.O-GlcNAc糖基化及相关催化酶在pre-B-ALL儿童白血病骨髓细胞中异常表达水平并影响Nalm-6细胞生物学行为1.1本研究共收集了 24例初发pre-B-ALL患儿和10例健康儿童的骨髓标本,两组的其年龄、性别的差别无统计学意义;采用免疫磁珠分选BM-MNCs的CD19+高达 90.47%。1.2和健康儿童组相比,B-ALL患儿组整体O-GlcNAc糖基化水平(p<0.01)及OGT明显升高(p<0.001)、OGA的蛋白表达水平降低(p<0.001),差异均具有统计学意义。1.3 合成了 OGT-siRNA 及 NC-siRNA,以 OGT-siRNA 转染 48h 后 Western-Bloting结果显示OGT的表达以及O-GlcNAc糖基化水平均明显下调,表明干扰其表达有效。以不同的Alloxan作用于Nalm-6细胞24h后,Western-Bloting技术结果显示O-GlcNAc糖基化水平、OGT表达水平逐渐降低,证明Alloxan可以用作OGT的抑制剂。1.4 CCK-8细胞增殖实验显示OGT-siRNA和Alloxan处理后Nalm-6细胞增殖明显减慢,差异均有统计学意义。1.5细胞凋亡实验显示:Alloxan可以诱导Nalm-6细胞凋亡[Alloxan组vs对照组(15.19±2.539)%对(21.91±4.105)%,P<0.05];同样的,OGT-siRNA 转染后Nalm-6细胞也出现凋亡[OGT-siRNA组vs对照组(21.72 ± 1.903%)对(11.07± 1.069%),p<0.05]。2.O-GlcNAc糖基化通过PI3K/Akt/c-Myc通路在Nalm-6细胞糖酵解过程的调控作用。2.1 LY294002可导致Nalm-6细胞中磷酸化的Akt(Akt Ser473/Akt)、磷酸化c-Myc(c-Myc Thr58/c-Myc)表达量明显下调,表明c-Myc是PI3K/Akt下游分子,LY294002可以抑制该通路的活性。2.2 Western-Blotting 法结果显示 H-LDH 患儿组的 CD19+BM-MNC 比 L-LDH 患儿组PI3K蛋白表达上调,Akt及c-Myc的磷酸化程度均上调,提示H-LDH患儿组中存在PI3K/Akt/c-Myc过度激活。2.3 Seahorse XF24细胞能量分析仪检测ECAR结果显示LY294002处理后的Nalm-6细胞的胞外酸化率ECAR及各项统计指标的发生明显变化,提示PI3K/Akt/c-Myc通路的抑制可下调胞内糖酵解水平。2.4临床样本分析结果:与L-LDH患儿相比,H-LDH患儿中O-GlcNAc糖基水平更高,OGT表达增高,但OGA的表达并未有明显差别。2.5糖酵解活跃程度检测:ECAR结果显示,与control组相比,OGT-siRNA组的ECAR水平及相关指标均明显下调,而组和NC-siRNA组无明显差异;2.6糖酵解催化过程相关分子表达:实时荧光定量PCR结果显示干扰OGT表达可以下调 HK2、HIF-1α、LDHA、PFK1 的 mRNA 表达(p<0.05),但 Glut1 无明显变化。2.7 PI3K/Akt/c-Myc通路激活情况:干扰OGT表达后PI3K的蛋白表达下调,Akt、c-Myc的磷酸化水平均下调,提示干扰OGT可以抑制PI3K/Akt/c-Myc通路激活。2.8糖酵解活跃程度检测:OGT-siRNA+IGF-1组较OGT-siRNA组糖酵解水平升高,表明激活PI3K/Akt通路可以拮抗干扰OGT所致的糖酵解抑制作用。3.联合应用糖酵解抑制剂2-DG及靶向抑制OGT可增强对Nalm-6杀伤效果3.1 Western-Blotting结果显示经过2-DG处理后,Nalm-6细胞O-GlcNAc糖基化及OGT逐渐下调。3.2 CCK-8结果显示:2-DG+OGT-siRNA组的OD值明显低于其他3组,说明联合应用2-DG及靶向抑制OGT可以加强对Nalm-6细胞增殖抑制的影响。3.3细胞凋亡实验结果示2-DG+OGT-siRNA组的细胞凋亡比例明显高于其他3组,差异有统计学意义。结论:1.在pre-B-ALL患儿的CD19+BM-MNC中存在过度的O-GlcNAc糖基化修饰以及关键催化酶OGT表达的升高,尤其是在高血清LDH的患儿中。2.干扰OGT从而降低O-GlcNAc修饰水平可以降低PI3K/Akt/c-Myc通路中重要信号蛋白的磷酸化水平,进而影响细胞糖酵解的代谢过程。3.靶向糖酵解过程关键酶、干扰抑制OGT的表达及两者联用均可以阻遏Nalm-6细胞的增殖,促进凋亡,这对于儿童白血病的治疗有极其重要的理论指导意义。更多还原

【Abstract】 Background:Precursor B-cell lymphoblastic leukemia(pre-B-ALL)is a common malignant disease in children.Although the current long-term disease-free survival rate increased significantly,but there are still 20%-30%of children eventually died,the reason lies in the process of chemotherapy without remission and recurrence.In recent years,there have been a lot of reports on the regulation of nucleic acid and protein levels in pre-B-ALL and their development.However,few reports on glycosylation abnormalities have been reported,especially about O-GlcNAc glycosylation.O-GlcNAc glycosylation is an important protein post-translational modification,first discovered by Gerald W.Hart.In recent years,it has been found that there are hundreds of post-translational modifications of proteins,of which glycosylation,especially O-GlcNAc glycosylation is particularly noticeable.O-GlcNAc glycosylation is a dynamically reversible process in whichβ-N-acetylglucosaminyltransferase(OGT)is responsible for glycosylation andβ-Nacetylglucosaminidase(OGA)is responsible for glycosylation removal.In vivo,O-GlcNAc glycosylation controls basic life processes such as protein synthesis,chromatin structure,demethylation of DNA and biological circadian patterns.Some signaling proteins or transcription factors such as Akt,c-Myc,p53 and NF-κB are glycosylated by O-GlcNAc.In recent years,a large number of literatures have proved that O-GlcNAc glycosylated protein plays an important role in various cancers,such as breast cancer,prostate cancer and bladder cancer,but not in pre-B-ALL and its drug resistance.In this study,bone marrow specimens of pre-B-ALL children patients and pre-B-ALL cell line Nalm-6 cells were selected as the main research objects.From the perspective of glycogen biology,the roles of O-GlcNAc glycosylation and related enzymes OGT and OGA in the development and drug resistance of pre-B-ALL were analyzed,and its mechanism from the perspective of cellular glycolytic metabolism was explored.Objective:1.To investigate the expression of O-GlcNAc glycosylation and related enzymes in pre-B-ALL patients leukemia cells and the effect of abnormal O-GlcNAc glycosylation on the biology of Nalm-6 cells;2.To investigate the regulatory role of abnormal O-GIcNAc glycosylation in the glycolysis of Nalm-6 cells via the PI3K/Akt/c-Myc pathway.3.To investigate the effect of combined use of glycolysis inhibitor 2-DG and targeted inhibition of OGT on the killing effect of Nalm-6.Methods:1.To investigate the expression of O-GlcNAc glycosylation and related enzymes in pre-B-ALL patients leukemia cells and the effect of abnormal O-GlcNAc glycosylation on the biology of Nalm-6 cells;1.1 Bone marrow specimens were collected from newly diagnosed pre-B-ALL patients at from September 2015 to September 2016.Specimens were collected using heparin anticoagulation tubes.Immediately after the specimen was collected,lymphocytes were separated and bone marrow mononuclear cells(BM-MNCs)were separated using lymphocyte separation fluid by density gradient centrifugation.CD19+B lymphocytes were sorted by immunomagnetic beads.The purity of the transfected cells was detected by flow cytometry.1.2 The overall O-GlcNAc glycosylation and OGT,OGA protein expression levels in pre-B-ALL children and healthy children were detected by Western-Blotting.1.3 The acute pre-B lymphocyte leukemia cell line Nalm-6 cells were used as the research object,and the biological behavior of Nalm-6 cells was detected by the RNA interference technology and Alloxan,an OGT specific inhibitor.The RNA interference method:siRNAs for OGT genes(OGT-siRNA)and NC-siRNAs for negative controls were synthesized.OGT-siRNA and NC-siRNA were transfected into Nalm-6 cells by Lipofectamine2000.After 48h,the expression of OGT and the level of O-GlcNAc glycosylation were detected by Western-Bloting to detect the interference effect.Inhibitor method:Nalm-6 cells treated with Alloxan for 24h,Western-Bloting was used to detecte O-GlcNAc glycosylation levels,OGT expression levels to verify the inhibition;After the inhibitory effect was verified,the effect on the biological behavior was subsequently examined after exposure to Nalm-6 cells with Alloxan at a concentration of 10mmol/L.1.4 Cell proliferation:The absorbance at 450 nm was detected by CCK-8 at 24h,48h,72h,and 96h after OGT inhibition(OGT-siRNA or Alloxan)and the corresponding proliferation curve was drawn.1.5 Cell apoptosis:Annexin V-PI double staining method was used to detect apoptosis ratio after OGT inhibition(OGT-siRNA 48h or Alloxan 24h)by flow cytometry.2.To explore the regulatory role of abnormal O-GlcNAc glycosylation in the glycolysis of Nalm-6 cells via the PI3K/Akt/c-Myc pathway.2.1 To verify that c-Myc is regulated by the PI3K/Akt pathway:Nalm-6 cells were treated with LY294002(PI3K/Akt pathway inhibitor)at different concentrations(0,1,5,and 10 μmol/L),the protein expression of Akt,phosphorylated Akt(Ser473),c-Myc,and phosphorylated c-Myc(Thr58)were detected by Western-Blotting.2.2 To compare activation of PI3K/Akt/c-Myc in CD 19 + cells and serum lactate dehydrogenase(H-LDH)children with ALL with low serum lactate dehydrogenase(L-LDH):Compared with 400u/L as the cut-off point to distinguish L-LDH group and H-LDH group,the detection method is the same as 2.1.2.3 Activity test of glycolysis:Nalm-6 cells were treated with different concentrations of LY294002(0,1,5,10p μmol/L)for 48h.The extracellular acidification rate(ECAR)was measured by seahorse XFe24 cell energy analyzer.The changes of baseline,glycolytic capacity,and glycolytic reserve were statistically analyzed.2.4 Analyse O-GlcNAc glycosylation levels and OGT,OGA expression in leukemia children with L-LDH and H-LDH level.2.5 Activity test of glycolysis:Nalm-6 cells were divided into three groups:the experimental group,transfected with OGT-siRNA group,called the OGT-siRNA group;the negative control group,ransfected with negative control siRNA,called the NC-siRNA group;the blank control group,transfected with only Lipofectamine2000.ECAR detection of the above three groups,the specific method with 4.3.2.6 Relative molecular detection of glycolysis catalysis:The mRNA expression levels of HK2,Glutl,HIF-1α,LDHA and PFK1 in the above three groups were detected by real-time fluorescence quantitative PCR,and β-actin was taken as the internal control for statistical analysis.2.7 Detection of PI3K/Akt/c-Myc pathway activation:detect the phosphorylation of key molecules in the pathway in the above three groups.The detection is the same as 2.1.2.8 The effect of IGF-1 on OGT-siRNA-induced glycolysis inhibition:OGT-siRNA+ IGF-1 group:Nalm-6 cells were treated by OGT-siRNA and IGF-1 simultaneously for 48h.OGT-siRNA group:Nalm-6 cells were treated with only OGT-siRNA for 48h.In the control group,only Lipofectamine2000 and an equal amount of PBS were added.The glycolytic test assay was performed as described in 4.3.3.To investigate the effect of combined use of glycolysis inhibitor 2-DG and targeted inhibition of OGT on the killing effect of Nalm-6.3.1 The effect of 2-DG on O-GlcNAc glycosylation and OGT in Nalm-6 cells:The effects of 2-DG at different concentrations(0,2.5,5mmol/L)on the O-GlcNAc glycosylation and OGT protein expression were examined by Western-Blotting.3.2 The effect of 2-DG on OGT expression and the proliferation of Nalm-6 cells:The OD value of 450 nm of OGT-siRNA transfected Nalm-6 cells at 48,72,and 96 h was detected by CCK-8 method.3.3 To examine the effect of 2-DG combined with targeted inhibition of OGT on the proliferation of Nalm-6 cells:Nalm-6 cells were divided into three groups:The 2-DG group was treated with 2-DG(2.5 mmol/L);The 2-DG + NC group was treated with 2-DG(2.5 mmol/L)and NC-siRNA;The 2-DG + OGT-siRNA group was treated with 2-DG(2.5 mmol/L)and OGT-siRNA.The proliferation detection method is the same as 1.4.Results:1.Abnormal expression of O-GlcNAc glycosylation and related catalytic enzymes in myeloid cells of pre-B-ALL and influence the biological behavior of Nalm-6 cells1.1 In our study,24 bone marrow samples from 24 newly diagnosed pre-B-ALL children and 10 healthy children were collected.There was no significant difference in age and sex between the two groups.The CD19+ BM-MNCs was up to 90.47%sorted by immunomagnetic beads.1.2 Compared with healthy children,the overall O-GlcNAc glycosylation levels(P<0.01)and OGT(P<0.001)and OGA protein expression levels(P<0.001)in children with B-ALL were significantly lower.Differences were statistically significant.1.3 OGT-siRNA and NC-siRNA were successfully synthesized.Western Bloting results showed that the expression of OGT and the level of O-GlcNAc glycosylation were significantly down-regulated after OGT-siRNA transfection for 48 h,indicating that interfering with the expression of OGT was effective.1.4 Western blotting showed that O-GIcNAc glycosylation level and OGT expression level decreased gradually after Alloxan treatment on Nalm-6 cells for 24h.Alloxan could be used as an inhibitor of OGT.1.5 The CCK-8 proliferation assay showed that the proliferation of Nalm-6 cells was significantly slowed down after OGT-siRNA or Alloxan treatment.The differences were statistically significant.1.6 The analyse of apoptosis showed Alloxan induced the apoptosis of Nalm-6 cells[Alloxan group vs control group(15.19±2.539)%vs(21.91±4.105)%,P<0.05].Similarly,Nalm-6 cells also showed apoptosis[OGT-siRNA group vs control group(21.72 ± 1.903%vs 11.07 ± 1.069%),p<0.05].2.Abnormal O-GlcNAc Glycosylation regulates the Nalm-6 cell glycolytic process via the PI3K/Akt/c-Myc pathway.2.1 LY294002 resulted in a significant downregulation of phosphorylated Akt(Akt Ser473/Akt)and phosphorylated c-Myc(c-Myc Thr58/c-Myc)in Nalm-6 cells,indicating that c-Myc is a downstream PI3K/Akt molecule,LY294002 can inhibit the activity of this pathway.2.2 The results of Western-Blotting showed that the expression of PI3K protein and the phosphorylation of Akt and c-Myc in CD19+ BM-MNC group were higher than those in L-LDH group,which suggested that there is over-activation of PI3K/Akt/c-Myc pathway in children with H-LDH.2.3 EACR test reults using Seahorse XF24 cell energy analyzer showed that NLY294002 could lead to significant changes in the extracellular acidification rate of Nalm-6 cells and the changes of various statistical indicators,suggesting that PI3K/Akt/c-Myc pathway can down regulate intracellular glycolysis level.2.4 Analysis of Western-Blotting results of clinical samples:Compared with children with L-LDH,O-GlcNAc glycosyl levels were higher in children with H-LDH children,OGT expression was increased,but OGA expression was not significantly different.2.5 The results of glycolytic activation showed that the level of ECAR and related indicators in OGT-siRNA group were significantly decreased compared with the control group,but there was no significant difference between the control group and the NC-siRNA group.2.6 The results of real-time PCR showed that the expression of HK-2,HIF-1α,LDHA and PFK1 were down-regulated by interfering OGT(p<0.05),but Glutl had no significant change.2.7 Activation of PI3K/Akt/c-Myc pathway:The protein expression of PI3K was down-regulated and the phosphorylation levels of Akt and c-Myc were down-regulated after OGT was interfered,suggesting that OGT interfered with the activation of PI3K/Akt/c-Myc pathway.2.8 The results of glycolytic activation showed that compared with OGT-siRNA group,the level of glycolysis in OGT-siRNA + IGF-1 group was increased,indicating that activation of PI3K/Akt pathway can antagonize the inhibition of OGT-induced glycolysis inhibition.3.Combination of glycolysis inhibitor 2-DG and targeted inhibition of OGT can enhance the killing effect on Nalm-63.1 Western-Blotting results showed that O-GlcNAc glycosylation and OGT in Nalm-6 cells gradually decreased after 2-DG treatment.3.2 The results of CCK-8 showed that the proliferation of Nalm-6 cells gradually decreased after interfering with OGT expression.3.3 The results of CCK-8 showed that the OD value of 2-DG + OGT-siRNA group was significantly lower than the other 3 groups,indicating that combined application of 2-DG and targeted inhibition of OGT can enhance the inhibition of proliferation of Nalm-6 cells.3.4 Cell apoptosis assay results showed that the proportion of apoptosis in 2-DG +OGT-siRNA group was significantly higher than the other 3 groups,the difference was statistically significant.Conclusion:1.Overexpression of O-GlcNAc and increased expression of the key catalytic enzyme OGT exist in CD19+ BM-MNC in pre-B-ALL children,especially in children with high serum LDH.2.Interfering with OGT to decrease the level of O-GIcNAc modification can reduce the phosphorylation of important signaling proteins in PI3K/Akt/c-Myc pathway and further affect the metabolic process of glycolytic.3.Targeting the key enzymes in glycolysis process,interfering with inhibiting the expression of OGT and their combination can both inhibit the proliferation of Nalm-6 cells and promote apoptosis,which is of extremely important theoretical significance for the treatment of childhood leukemia.更多还原