【作者】 胡艳妮；

【导师】 徐酉华；

【作者基本信息】 重庆医科大学 ， 儿科学， 2011， 博士

【摘要】 第一部分GSK-3β在Jurkat细胞株及儿童急性淋巴细胞白血病骨髓单个核细胞中的定位与表达目的检测GSK-3β在人急性淋巴细胞白血病细胞株Jurkat与儿童急性淋巴细胞（ALL）白血病骨髓单个核细胞（BMMNC）中的定位与表达水平。方法实验分为实验组（Jurkat细胞及ALL细胞），对照组（骨髓象正常的ITP），采用Real Time PCR技术、免疫荧光细胞化学染色及Westernblotting的方法，分别从mRNA和蛋白质水平进行检测。结果1.Real Time PCR结果显示，实验组与对照组均表达GSK-3βmRNA，Jurkat与ALL细胞中GSK-3β mRNA的表达水平分别为对照组的8.53和5.19倍，差异有统计学意义（P＜0.05）。2.免疫荧光细胞化学染色及Western blotting结果均显示，实验组细胞中GSK-3β定位于细胞浆及胞核，呈高表达；对照组细胞中GSK-3β仅在细胞浆呈中等度表达，细胞核中不表达。结论GSK-3β在Jurkat细胞株与ALL患儿BMMNC细胞核中均呈异常过度表达，提示GSK-3β发生了核转位，为GSK-3β调控细胞内信号分子打下了理论基础。第二部分抑制GSK-3β活性对急性淋巴细胞白血病细胞株Jurkat细胞凋亡的机制研究实验一GSK-3β抑制剂诱导急性淋巴细胞白血病细胞株Jurkat细胞凋亡的机制研究目的以Jurkat细胞作为实验细胞，采用GSK-3β抑制剂氯化锂（LiCl）和SB216763抑制GSK-3β，探讨抑制的GSK-3β介导的Wnt/β-catenin与NF-κB信号通路诱导细胞凋亡的分子机制。方法分别应用LiCl和SB216763抑制GSK-3β，①MTT法检测Jurkat细胞增殖，并绘制细胞生长曲线；②Annexin V-PE/7-AAD双染法结合流式细胞仪检测细胞凋亡；③Western blotting方法检测Jurkat细胞中GSK-3β及其下游信号分子β-catenin和NF-κB P65定位与表达水平的变化；④RT-PCR检测Wnt/β-catenin与NF-κB信号通路下游靶基因survivin、c-myc和cyclinD1表达水平的变化。结果1.在LiCl浓度≥20mM，SB216763浓度≥15μM时，Jurkat细胞生长受到明显抑制，并呈浓度-时间依赖性。2.以NaCl作为对照，LiCl在20、30、40mM浓度下的凋亡率分别为（10.89±1.32）%、（23.74±1.65）%、（38.26±4.37）%；以DMSO作为对照，SB216763各浓度组的细胞凋亡率分别为（14.71±2.53）%、（24.92±4.58）%、（43.25±4.72）%，实验组的细胞凋亡与对照组比较，差异均具有统计学意义（P＜0.05）。3.LiCl（20mM）抑制GSK-3β活性后，与对照组相比，β-catenin蛋白在胞浆和胞核中的表达分别增加了约2.15和1.63倍，差异有统计学意义（P＜0.05）；NF-κB P65蛋白在胞浆和胞核中的表达均无明显变化。SB216763(15μM)抑制GSK-3β活性后，与对照组相比，β-catenin蛋白在胞浆和胞核中的表达分别增加了约3.27和2.84倍，差异有统计学意义（P＜0.05）；NF-κB P65蛋白在胞浆和胞核中的表达也均无明显变化。4.下游靶基因survivin的表达明显下调，而c-myc与cyclin D1mRNA的表达水平无明显变化。结论GSK-3β抑制剂下调Jurkat细胞中GSK-3β的活性后，可减慢细胞增殖，促进细胞凋亡，推测其机制可能是GSK-3β抑制剂启动了其他的信号系统介导了Jurkat细胞的凋亡，而与β-catenin和NF-κB信号的关系不大。实验二外源性Wnt-3a对急性淋巴细胞白血病细胞株Jurkat细胞凋亡的机制研究目的以Jurkat细胞作为实验细胞，通过Wnt蛋白抑制GSK-3β活性，并激活Wnt/β-catenin信号通路，进一步探寻GSK-3β介导的Wnt/β-catenin信号通路诱导Jurkat细胞凋亡中的分子机制。方法应用基因重组技术构建的Wnt-3a重组腺病毒表达载体Ad5-Wnt-3a，采用重组腺病毒直接感染的方法对Jurkat细胞进行基因转染。实验分为三组：实验组（Jurkat/Ad5-Wnt-3a组）、空载体组（Jurkat/Ad5-GFP组）和空白对照组（Jurkat组）。①用荧光显微镜、流式细胞仪检测各组细胞转染率；②RT-PCR方法检测各组细胞内Wnt-3a mRNA的表达；③Western blotting检测细胞内β-catenin、GSK-3β蛋白的表达；④MTT法检测细胞增殖；⑤流式细胞仪法检测细胞周期；⑥Annexin V-PE/7-AAD双染法结合流式细胞仪检测细胞凋亡；⑦RT-PCR检测Wnt/β-catenin信号通路下游靶基因survivin、c-myc和cyclinD1的表达。结果1.将构建成功的重组腺病毒载体Ad5-Wnt-3a转染Jurkat细胞，筛选出最适转染滴度MOI＝60，转染率为(42.54±1.63)%。2.Wnt-3a转染后，实验组有效表达Wnt-3a基因；GSK-3β总蛋白的表达量显著下调；β-catenin蛋白表达量明显上调，为对照组的2.38倍；细胞增殖明显，增殖率约为21.2%，细胞G0/G1期下降，S期升高，未见明显细胞凋亡。3.RT-PCR检测结果显示实验组细胞内c-myc和cyclin D1mRNA表达分别为（0.75±0.04）和（0.75±0.03），相比空载体组，表达水平的增高具有统计学意义。而Survivin mRNA表达水平未见明显改变。结论腺病毒介导的Wnt-3a抑制Jurkat细胞中GSK-3β蛋白的表达，同时可激活细胞内经典Wnt/β-catenin信号途径，活化的β-catenin促进Jurkat细胞增殖与细胞周期改变，未见明显细胞凋亡，可能的机制是通过上调下游靶基因c-myc和cyclin D1的表达实现。第三部分抑制GSK-3β活性对儿童急性淋巴细胞白血病骨髓单个核细胞凋亡的机制研究目的以ALL患儿原代细胞为实验细胞，通过抑制剂氯化锂（LiCl）和SB216763抑制GSK-3β活性后，进一步探讨GSK-3β介导的Wnt/β-catenin与NF-κB信号通路诱导ALL细胞凋亡的分子机制。方法分别应用LiCl和SB216763抑制ALL细胞内GSK-3β，经过体外短期培养，①Western blotting方法检测ALL细胞中GSK-3β及其下游信号分子β-catenin与NF-κB p65的定位和表达水平的变化；②凝胶迁移率实验（EMSA）检测核转录因子NF-κB的DNA结合活性；③AnnexinV-PE/7-AAD双染法结合流式细胞仪检测细胞凋亡；④RT-PCR检测Wnt/β-catenin与NF-κB信号通路下游靶基因survivin、c-myc和cyclinD1表达水平的变化。结果1.ALL细胞经GSK-3β抑制剂LiCl和SB216763处理后，GSK-3β在细胞浆中的蛋白水平未见明显变化，而其胞核蛋白表达量明显下降；β-catenin蛋白在细胞浆中的表达量显著增加，而转位入核的β-catenin表达略有增加；核转位蛋白NF-κB P65在胞浆/胞核中均无明显变化，但核内NF-κB P65的DNA结合活性明显降低。2.SB216763浓度梯度增加（5、10、15μM）的实验组细胞凋亡率随之升高，分别为（36±5）%、（52±7）%和（70±4）%，LiCl在其细胞亚毒性浓度内（5、10mM），诱导ALL细胞的凋亡呈浓度依赖性，与对照组相比均有统计学意义。3.对照组细胞经GSK-3β抑制剂LiCl和SB216763处理后，发现对照组细胞的凋亡率为15%~20%，未受到明显影响。但Western blotting结果显示转位入核的β-catenin蛋白表达显著增加。4.RT-PCR结果显示survivin mRNA的表达水平明显下降，c-myc和cyclin D1的表达水平未见明显变化。结论1.以ALL原代细胞作为实验细胞，抑制其GSK-3β活性后，并不改变核转位蛋白NF-κB P65在胞浆/胞核中的表达，却下调NF-κB P65的转录活性，并通过下调抗凋亡基因survivin的表达而促进ALL细胞的凋亡；β-catenin的表达水平增加并未明显影响细胞的凋亡与增殖，其机理有待进一步的研究。2.对照组细胞经GSK-3β抑制剂处理后，细胞凋亡未受明显影响，推测可能与Wnt/β-catenin信号的活化有关。更多还原

【Abstract】 PART ONE THE LOCALIZATION AND EXPRESSIONOF GSK-3Β IN JURKAT CELL LINE AND BONEMARROW MONONUCLEAR CELLS OF CHILDHOODACUTE LYMPHOBLASTIC LEUKEMIAObjectives: To investigate the localization and expression of GSK-3βin Jurkat cells and bone marrow mononuclear cells (BMMNC) of childhoodacute lymphoblastic leukemia (ALL).Methods: The study was divided into the experimental group (Jurkatcell line and ALL cells) and the control group (ITP with normal bonemarrow). The mRNA and protein levels of GSK-3β were detected by RealTime PCR, immunofluorescence staining and Western blotting methods,respectively.Results:1. Real Time PCR analysis showed that both experimental andcontrol groups expressed GSK-3β mRNA. The expression levels of GSK-3βmRNA in Jurkat and ALL cells were8.53and5.19-fold compared to of the control group (P＜0.05).2. Immunofluorescence staining and Western blotting results showedthat GSK-3β was highly expressed in the cytoplasm and the nucleus of theexperimental group cells; in the control group, GSK-3β was only expressedin the cytoplasm but not expressed in the nucleus.Conclusions: GSK-3β was abnormally over-expressed in the nucleusof Jurkat cells and BMMNC of children with ALL. Nuclear accumulation ofGSK-3β seems to lay a theoretical foundation to regulate the intracellularsignaling molecules. PART TWO THE INHIBITION OF GSK-3ΒACTIVITY INDUCEED CELL APOPTOSIS OF ACUTELYMPHOBLASTIC LEUKEMIA CELL LINE JURKATEXPERIMENT A GSK-3Β INHIBITORS INDUCEDCELL APOPTOSIS IN JURKATObjectives: To explore the molecular mechanisms of apoptosisinduced by GSK-3β mediated Wnt/β-catenin and NF-κB signaling pathwayin Jurkat cells treated with GSK-3β inhibitors lithium chloride (LiCl) and SB216763.Methods: Jurkat cells were treated with LiCl and SB216763,respectively. Cell proliferation was detected by MTT assay to make the cellgrowth curve; Cell apoptosis was detected by Annexin V-PE/7-AAD doublestaining combined with flow cytometry; The localization and expression ofGSK-3β and its downstream signaling molecules β-catenin and NF-κB P65in Jurkat cells were detected by Western blotting; The changes ofdownstream target gene (survivin, c-myc and cyclinD1) expressions inWnt/β-catenin and NF-κB signaling pathways were detected by RT-PCR.Results:1.In the concentration of LiCl≥20mM, SB216763≥15μM, cellgrowth was obviously inhibited in a concentration-time-dependent manner.2. The mean number of Jurkat cells treated with LiCl at20,30,40mMconcentrations reached (10.89±1.32)%,(23.74±1.65)%,(38.26±4.37)%(NaCl as control, P＜0.05); The mean number of Jurkat cells treated withSB216763at different concentrations reached (14.71±2.53)%,(24.92±4.58)%,(43.25±4.72)%(DMSO as control, P＜0.05).3. Jurkat cells were treated with20mM LiCl, compared with the controlgroup, the expression of β-catenin protein in the cytoplasm and nucleusincreased by2.15and1.63-fold (P＜0.05); The expression of NF-κB P65protein in the cytoplasm and the nucleus had no obvious changes. WhileJurkat cells were treated with15μM SB216763, compared with the controlgroup, the expression of β-catenin protein in the cytoplasm and nucleus increased by3.27and2.84-fold (P＜0.05); The expression of NF-κB P65protein in the cytoplasm and nuclei had no significant changes.4. The expression of survivin mRNA was significantly reduced(P＜0.05), while the levels of c-myc and cyclin D1mRNA expression did notchange.Conclusions: GSK-3β inhibitors may slow down cell proliferation andpromote apoptosis of Jurkat cells. The mechanism may be GSK-3βinhibitors induced apoptosis in Jurkat cells by activated other signal systems,but not by β-catenin and NF-κB signaling pathways. EXPERIMENT B EXOGENOUS WNT-3A FOR CELLAPOPTOSIS IN JURKATObjectives: To explore the molecular mechanisms of apoptosisinduced by GSK-3β inhibition mediated Wnt/β-catenin and NF-κB signalingpathway in Jurkat cells through recombinant adenovirus vector-mediatedWnt-3a.Methods: Using recombinant DNA technology to build the Wnt-3arecombinant adenovirus expression vector Ad5-Wnt-3a. Jurkat cells weretransfected by the direct infection of recombinant adenovirus. Jurkat cellswere divided into three groups: the experimental group (Jurkat/Ad5-Wnt-3a group), the empty vector group (Jurkat/Ad5-GFP group) and the blankcontrol group (untransfected Jurkat group). The transfection or infectionefficiencies of cells in every group were detected by fluorescence and flowcytometry. The expression level of Wnt-3a mRNA in every group wasdetected by RT-PCR; The protein expression of β-catenin and GSK-3β wasdetected by Western blotting; Cell proliferation and cell cycle were detectedby MTT assay and by flow cytometry, respectively; Cell apoptosis wasmeasured by Annexin V-PE/7-AAD double staining combined with flowcytometry; The changes of downstream target gene (survivin, c-myc andcyclinD1) expression in Wnt/β-catenin signaling pathway were detected byRT-PCR.Results:1. Jurkat cells were transfected successfully by recombinedadenovirus Ad5-Wnt-3a, with MOI=60for the optimal transfection titer,and the transfection rate was (42.54±1.63)%.2. The experimental group expressed Wnt-3a gene efficiently aftertransfection of Ad5-Wnt-3a. The total protein expression of GSK-3βwassignificantly reduced; β-catenin protein expression was significantlyincreased2.38times compared with the control group; Actived β-catenincould significantly promote the proliferation of Jurkat cells (proliferationrate was about21.2%), and cause G0/G1phase cells down, S phase cellsincreased, but cell apoptosis was not obvious. There was no significant cellapoptosis. 3. RT-PCR results showed that the c-myc and cyclin D1mRNAexpression were (0.75±0.04) and (0.75±0.03) in the experimental group,significantly increased compared with empty vector group. The expressionof survivin mRNA did not change significantly.Conclusions: The exogenous Wnt-3a gene mediated by recombinantadenovirus vector could downregulate GSK-3β activity and activate thecanonical Wnt/β-catenin signaling pathway in Jurkat cell line. The activationof β-catenin promoted cell proliferation and cell cycle progression, but didnot affect cell apoptosis. Its mechanism may be β-catenin regulated theexpression s of downstream target genes c-myc and cyclin D1. PART THREE THE INHIBITION OF GSK-3ΒACTIVITY INDUCED CELLS APOPTOSIS IN CHILDRENWITH ACUTE LYMPHOBLASTIC LEUKEMIAObjectives: To explore the molecular mechanisms of apoptosisinduced by GSK-3β mediated Wnt/β-catenin and NF-κB signaling pathwayin ALL cells treated with GSK-3β inhibitors LiCl and SB216763.Methods: ALL cells were treated with LiCl and SB216763in vitroshort-term culture. The changes of GSK-3β and the localization andexpression level of the signaling molecules β-catenin and NF-κB P65were detected by Western blotting; Electrophoretic mobility shift assay (EMSA)was used to assess the binding of nuclear transcription factor NF-κB activityto the DNA; Annexin V-PE/7-AAD double staining combined with flowcytometry to detect cell apoptosis; Further, the changes of downstream targetgene (survivin, c-myc and cyclinD1) expression in Wnt/β-catenin andNF-κB signaling pathways was detected by RT-PCR.Results:1.ALL cells were treated with GSK-3β inhibitors LiCl andSB216763, the protein level of GSK-3β did not change in the cytoplasm, butits nuclear protein expression decreased significantly; The signaling criticaleffect molecular β-catenin protein expression in Wnt/β-catenin pathwayincreased significantly in the cytoplasm, while β-catenin expressiontranslocated into the nucleus increased slightly; The nuclear translocationprotein NF-κB P65in NF-κB signaling pathway was no significant changesin the cytoplasm/nucleus of cell, but the DNA binding activity of nuclearNF-κB P65was significantly reduced.2. SB216763concentration gradient increased (5,10,15μM) in theexperimental group, with an increasing apoptosis rate (36±5)%,(52±7)%and (70±4)%, respectively; LiCl in the cell sub-toxic concentrations with(5,10mM), induced apoptosis in ALL cells in a concentration dependentmanner.the control group were statistically significant.3. The apoptosis was not significantly affected and found to be15%to20%in the control cells with LiCl or SB216763treatment. However, Western blotting showed that expression of β-catenin protein translocatedinto the nucleus was significantly increased.4. RT-PCR showed that the downstream anti-apoptosis gene survivinexpression was reduced, but c-myc and cyclin D1mRNA expression levelsdid not change.Conclusions: The inhibition of GSK-3β activity in ALL primary cellsdid not change the cytoplasm/nucleus expression of the nuclear translocationprotein NF-κB P65, but reduced NF-κB P65transcriptional activity, whichpromoted apoptosis of ALL cells by decreasing the expression ofanti-apoptotic gene survivin. The level of β-catenin expression did notsignificantly affect cell apoptosis and proliferation, and the mechanismneeds further study. Apoptosis was not significantly affected in control cellstreated with GSK-3β inhibitors, suggesting that it may be associated withWnt/β-catenin signal activation.更多还原