靶向抑制GSK-3β介导的Wnt与NF-κB信号通路在急性淋巴细胞白血病细胞凋亡中的机制研究
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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。
PART ONE THE LOCALIZATION AND EXPRESSIONOF GSK-3Β IN JURKAT CELL LINE AND BONEMARROW MONONUCLEAR CELLS OF CHILDHOODACUTE LYMPHOBLASTIC LEUKEMIA
Objectives: 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 JURKAT
EXPERIMENT A GSK-3Β INHIBITORS INDUCEDCELL APOPTOSIS IN JURKAT
Objectives: 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 JURKAT
Objectives: 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 LEUKEMIA
Objectives: 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.
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。
PART ONE THE LOCALIZATION AND EXPRESSIONOF GSK-3Β IN JURKAT CELL LINE AND BONEMARROW MONONUCLEAR CELLS OF CHILDHOODACUTE LYMPHOBLASTIC LEUKEMIA
Objectives: 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 JURKAT
EXPERIMENT A GSK-3Β INHIBITORS INDUCEDCELL APOPTOSIS IN JURKAT
Objectives: 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 JURKAT
Objectives: 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 LEUKEMIA
Objectives: 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.
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