【作者】 顾金海；

【导师】 李刚； 孙涛；

【作者基本信息】 山东大学 ， 神经外科， 2008， 博士

【摘要】 人脑胶质瘤是中枢神经系统中最为常见的原发性肿瘤,其中的高级别胶质瘤呈浸润性生长,手术、放疗、化疗等均难根治,患者的平均生存期仅1年左右。因此,探寻新的具有显著疗效的胶质瘤治疗方法始终是神经外科关注的焦点之一。迄今为止,脑胶质瘤基因治疗研究仍然没有取得人们所期望的重大突破,主要是因为以往的基因治疗方法,往往是针对某一个癌相关基因进行干预,但肿瘤的发生与发展却是一个多种基因参与、多环节、多阶段、多步骤的复杂过程,仅仅通过抑制某个癌相关基因或补充某个抑癌基因来达到治愈的目的是很难实现的,新的希望只能寄托于多靶点、多策略的综合基因治疗。近年来,研究者们已经认识到某些关键的转录因子,通过相应的细胞信号传导通路,同时掌控着下游多个癌相关基因或凋亡相关基因的表达,形成了影响肿瘤发生、发展、扩散等生物学特性的相关基因启闭的“总阀门”。特异性抑制这些关键核转录因子,就可以引发相关基因“级联式”或“瀑布式”的组合阻断,从而达到一种药物阻断多种癌基因表达的综合治疗效应。因此,针对癌症相关转录因子进行干预,在当前已经成为包括人脑胶质瘤在内的多种恶性肿瘤基因治疗的研究热点。信号传导和转录活化因子3（STAT3,Signal Transducers and Activators ofTranscription 3）是细胞信号传导通路JAK2/STAT3途径的关键转录因子,掌控着下游bcl-xl、bcl-2、c-myc、cyclinD1、survivin、mcl-1和VEGF等多种基因的转录激活,对调节细胞的生长、分化和凋亡等过程发挥着重要的影响。异常的STAT3信号通路参与了包括胶质瘤在内的多种肿瘤的恶性转化和增殖,通过上调多种致癌基因的表达,抑制肿瘤细胞的凋亡,促进肿瘤细胞的增殖,最终导致恶性肿瘤的发生和发展,被确定为一种重要的转录因子类癌基因。研究表明,靶向性阻断STAT3信号通路,可以有效下调胶质瘤细胞中多种致癌基因的表达水平,明显抑制肿瘤细胞增殖。STAT3是胶质瘤基因治疗研究中非常有效的分子靶点。诱骗寡核苷酸技术（decoy oligodeoxynucleotide,decoy ODN）,将体外人工合成、有高度亲和性,并且模拟目标转录因子靶基因上启动子顺式元件的序列合成的小分子“诱骗”DNA,转染导入肿瘤细胞,竞争性地抑制靶基因启动子与转录因子的结合,降低其转录活性,阻止这些致癌基因的转录激活,从而诱导凋亡,抑制肿瘤细胞增殖。相对而言,decoy ODN策略具有效率高、成本低、特异性强、作用广泛、治疗靶标丰富等优点,是肿瘤基因治疗的一条有效途径,目前已经被广泛地应用于多项实验和临床研究。研究结果证实,应用decoy ODN靶向性阻断STAT3信号通路,可以有效降低癌基因cyclinD1、c-myc、survivin、VEGF、bcl-2、bcl-xl等转录和翻译的表达水平,诱导凋亡,抑制肿瘤细胞增殖,治疗头颈部鳞状上皮细胞癌、乳腺癌和肺癌等恶性肿瘤。本课题旨在研究和探讨应用decoy ODN靶向性阻断STAT3信号通路抑制人脑胶质瘤细胞增殖的效应和机制。研究内容分为三个部分。一、人脑胶质瘤组织细胞中STAT3异常表达研究目的检测人脑胶质瘤细胞系以及临床标本中STAT3及其磷酸化活化蛋白的表达情况,研究人脑胶质瘤中STAT3的异常表达及持续活化的意义,为针对STAT3治疗胶质瘤的实验研究奠定坚实的基础。方法1、Western印迹方法检测24例人脑胶质瘤标本中STAT3及其磷酸化活化蛋白的表达,并对其异常表达及持续活化的情况进行分析。2、Western印迹方法检测人脑胶质瘤细胞系U251和A172中STAT3及其磷酸化活化蛋白的表达。结果1、人脑胶质瘤的临床标本中广泛存在着STAT3和磷酸化STAT3的异常表达。与正常人脑组织相比,胶质瘤组织中存在着STAT3的过高表达和持续活化,其中,酪氨酸磷酸化的表达阳性率为75%（24例中有18例）,丝氨酸磷酸化的表达阳性率为66.67%（24例中有16例）。2、人脑胶质瘤细胞系U251和A172中存在STAT3和磷酸化STAT3的异常表达。两种细胞系中存在STAT3和它的两种活化形式（705位酪氨酸磷酸化STAT3与727位丝氨酸磷酸化STAT3）的表达,其中尤其以酪氨酸磷酸化明显。结论1、人脑胶质瘤组织和细胞中广泛存在着STAT3的过高表达及持续活化,说明STAT3信号传导通路在胶质瘤中具有重要意义,为抑制STAT3治疗胶质瘤的研究奠定了坚实的基础。2、U251和A172中存在STAT3和磷酸化STAT3的异常表达,可以作为本课题的研究对象。二、Decoy ODN靶向性阻断STAT3信号通路抑制人脑胶质瘤细胞增殖研究目的研究阳离子脂质体转染STAT3 decoy ODN的效果和效率、decoy ODN靶向性阻断STAT3信号通路的特异性以及应用decoy ODN靶向性阻断STAT3信号通路对人脑胶质瘤细胞增殖的抑制效应。方法1、应用阳离子脂质体Lipofectamine^TM2000介导的DNA转染法将STAT3decoy ODN转染导入人脑胶质瘤细胞系U251和A172内。2、流式细胞术检测FITC标记的STAT3 decoy ODN在U251和A172中的转染效率。3、荧光显微镜观察FITC标记的STAT3 decoy ODN转染导入人脑胶质瘤细胞系U251和A172内的效果。4、荧光素酶报告法检测分析decoy ODN靶向性阻断STAT3信号通路的效果和特异性。5、细胞计数法检测decoy ODN靶向性阻断STAT3信号通路后人脑胶质瘤细胞增殖的抑制效应。6、MTT比色法检测decoy ODN靶向性阻断STAT3信号通路后人脑胶质瘤细胞增殖的抑制效应。7、[³H]-TdR掺入法检测decoy ODN靶向性阻断STAT3信号通路后人脑胶质瘤细胞增殖的抑制效应。结果1、应用阳离子脂质体Lipofectamine^TM2000介导的DNA转染技术,能够将STAT3 decoy ODN高效转染导入人脑胶质瘤细胞。流式细胞术检测结果显示,50 nmol/L FITC-STAT3 decoy ODN被Lipofectamine^TM2000介导转染后,U251细胞的MFI（Mean fluorescence intensity）是64.03%,A172细胞的MFI是56.22%。荧光显微镜观察结果直观显示,STAT3 decoy ODN被转染导入胶质瘤细胞,相当部分进入细胞核。2、Decoy ODN能够特异性抑制STAT3的转录活性,靶向性阻断STAT3信号通路。荧光素酶报告法结果表明,STAT3 decoy ODN能够抑制报告STAT3基因转录活性的荧光素酶的相对活性,而且这种抑制效应和其浓度成正比,25nmol/L和50 nmol/L的STAT3 decoy ODN对荧光素酶相对活性的抑制有统计学意义（P＜0.05）。作为对照,STAT3 decoy ODN并没有显示对报告STAT6基因转录活性的荧光素酶活性的抑制效应;Mutant control decoy ODN则不能对荧光素酶活性产生明显的抑制,不管是对STAT3,还是对STAT6。Western Blot检测结果显示,STAT3 decoy ODN转染24、48、72小时后,U251和A172细胞STAT3、酪氨酸磷酸化STAT3和丝氨酸磷酸化STAT3蛋白的表达并无显著变化。3、STAT3 decoy ODN能够明显抑制人脑胶质瘤细胞增殖。细胞计数结果显示STAT3 decoy ODN能够抑制胶质瘤细胞增殖,抑制效应和时间与转染ODN浓度相关。MTT比色法证实STAT3 decoy ODN对胶质瘤细胞增殖的抑制率与浓度和时间呈正相关。25和50 nmol/L的STAT3 decoy ODN与空白组（TE）比较有显著性差异（P＜0.05）,而Mutant control decoy ODN与其相比,差异则无意义（P＞0.05）。[³H]-TdR掺入实验显示,递增浓度的STAT3 decoy ODN干预U251和A172细胞48小时后,代表两种细胞DNA含量的CPM（Count per minute）值均随着decoy ODN转染浓度的升高而逐渐降低,25和50 nmol/L的与空白组（TE）比较有显著性差异（P＜0.05）;而Mutant control decoy ODN与其相比,差异则无意义（P＞0.05）。这些结果表明,STAT3 decoy ODN能明显胶质瘤细胞DNA的合成,再次证实STAT3 decoy ODN能够明显抑制人脑胶质瘤细胞增殖。结论1、应用阳离子脂质体Lipofectamine^TM2000介导的DNA转染技术,能够将STAT3 decoy ODN高效转染导入人脑胶质瘤细胞内,为其发挥阻断和抑制作用创造了良好的先决条件。2、STAT3 decoy ODN能够特异性地结合在STAT3分子上,有效抑制其转录活性,靶向性阻断STAT3信号通路。3、STAT3 decoy ODN能够安全有效地抑制人脑胶质瘤细胞增殖。三、Decoy ODN靶向性阻断STAT3信号通路抑制人脑胶质瘤细胞增殖的相关机制研究目的检测decoy ODN转染人脑胶质瘤细胞对其细胞周期和凋亡的影响,并对其相关基因的表达水平进行统计分析,进而深入探讨decoy ODN靶向性阻断STAT3信号通路抑制人脑胶质瘤细胞增殖的相关机制。方法1、流式细胞术检测decoy ODN靶向性阻断STAT3信号通路对U251和A172细胞周期的影响。2、Annexin/PI双染法检测decoy ODN靶向性阻断STAT3信号通路后U251和A172细胞的凋亡情况。3、RT-PCR方法检测分析STAT3 decoy ODN对细胞周期控制基因和凋亡相关基因mRNA表达水平的影响。4、Western印迹法检测分析STAT3 decoy ODN对细胞周期控制基因和凋亡相关基因蛋白表达水平的影响。结果1、STAT3 decoy ODN能够阻滞胶质瘤细胞从G1期向S期的转换。流式细胞术检测结果显示,50 nmol/L STAT3 decoy ODN干预24小时后,与对照组相比,两种胶质瘤细胞系处于G0/G1期细胞的百分比明显增多,U251细胞从49.58±1.09%增长至54.89±1.35%（P＜0.05）,A172从63.52±1.61%增长到72.03±1.14（P＜0.05）:同时处于S期细胞的比例明显减少,U251细胞从42.36±1.50%减少到37.89±0.67%（P＜0.05）,A172则从22.23±0.51%减少至16.00±0.40%（P＜0.05）。2、STAT3 decoy ODN能够诱导胶质瘤细胞凋亡。Annexin V/PI双染色法检测结果表明,与对照组相比,50 nmol/L STAT3 decoy ODN干预24小时后诱导的凋亡细胞,U251从5.44±0.50%增长至23.57±9.76%（P＜0.05）,A172从5.20±0.24%增长到22.48±0.85%（P＜0.05）,作为对照,Mutant control decoy ODN诱导的U251和A172凋亡细胞仅增加了大约6.81%和6.16%,无统计学意义（P＞0.05）。3、STAT3 decoy ODN能够下调细胞周期和凋亡相关基因的表达水平。RT-PCR检测结果显示,50 nmol/L STAT3 decoy ODN干预24小时后,U251细胞的c-myc、cyclinD1和bcl-xl的mRNA表达水平分别下调了52%、50%和27.67%,Al72分别下调46.67%、40.67%和28.87%,和对照组比较,有显著性差异（P＜0.05）。Western Blot检测结果显示,50 nmol/L STAT3 decoy ODN干预24小时后,U251细胞的cyclinD1和bcl-xl的蛋白表达水平分别下调了47.33%和85.66%,A172细胞分别下调38.25%和3.83%。因此,上述结果中,除了A172细胞的bcl-xl蛋白表达水平无显著性差异外,其他和空白对照、阳性对照组比较,均有统计学意义（P＜0.05）。结论1、STAT3 decoy ODN能够阻滞胶质瘤细胞从G1期向S期的转换。2、STAT3 decoy ODN能够诱导胶质瘤细胞凋亡。3、STAT3 decoy ODN能够下调细胞周期和凋亡相关基因的表达水平。总之,通过本研究表明,应用STAT3 decoy ODN能够下调胶质瘤细胞的细胞周期调控基因和凋亡基因的表达水平,通过阻滞胶质瘤细胞的细胞周期,诱导细胞凋亡,有效抑制人脑胶质瘤细胞的增殖。应用decoy ODN靶向性阻断STAT3信号通路,有望成为人脑胶质瘤基因治疗的一种新途径。更多还原

【Abstract】 Human glioma is the most common primary tumor of human central nervous system.The high-grade gliomas,which are highly invasive and aggressive tumors, have been resistant to conventional treating modalities including surgical resection, radiotherapy and chemotherapy.Despite the great advances made in the treatments recently,the median survival for patients with high-grade gliomas is still less than one year.Therefore,it has always been one of focus of neurosurgery to explore the novel and effective treatment.At present,dued mainly to some oncogenes been simply intervened in the previous studies,the gene therapy research for glioma has not gained the expected major breakthrough.Carcinogenesis is a multiple-link,multi-stage,and multi-step process,which involves in a great number of oncogenes and antioncogenes. So it is difficult to cure glioma only by intervening one cancer-related gene.The novel treatment will not benefit until it can simultaneously modulate various cancer-related genes’ expression.In recent years,it has been realized that some transcription factors, which is the key to modulate various cancer-related genes in the corresponding cell signaling pathway.The transcription factors can simultaneously and effectively modulate the transcription and translation of its downstream oncogenes,and it may be regarded as "general switch" that affect cancergenesis and aggravation.Through specific inhibition of the key transcription factor,a "cascade" or "waterfall"-like synthetic blockage to a variety of oncogenes will attain.Therefore it has gained great interests for target inhibition of transcription factor in studies of gene therapy for a variety of cancers,including human glioma and others.The transcription factor STAT3（signal transducer and activator of transcription 3） which controls the transcription activation of its downstream genes,including bcl-xl, bcl-2,c-myc,cyclinD1,survivin,mcl-1 and VEGF,is involved in the embyro development,physiologic regulation of cell growth,survival and differentiation,and pathogenesis of malignancies by participating in Jak2/STAT3 signaling pathway.Like many other malignancies,aberrant STAT3 activation was found in primary malignant gliomas and numerous cancer-derived cell lines.The constitutive activation of STAT3 contributes to the apoptosis inhibition and cell proliferation by up regulation of various oncogenes,which lead to the transformation and progression of glioma.So STAT3 has been defined as one of transcription factor-like oncogene.Experiments have showed that after STAT3 signaling pathway was significantly blocked,it could down-regulate the expression level of various oncogenes,and subsequently resulted in a depressed proliferation and increased apoptosis in human glioma cells.The result suggests that STAT3 acts as a promising molecular target of gene therapy for glioma.The decoy ODN strategy is based on the competiiion between the endogenous cis-element within the regulatory regions of target genes promoters and exogenously added molecules mimicking the specific cis-elements.Transfection of STAT3 decoy ODN,a small molecule DNA synthesized in vitro and with high compatibility,into cells will competitively inhibit the binding of STAT3 with its endogenous ciselements, prevent the transcription and activation of the oncogene,and subsequent result in cells apoptosis and inhibition of glioma cells proliferation.Because decoy ODN strategy is better than other approaches in the aspect of efficiency,cost,and specificity,it has been widely used as a novel and effective approach in a number of laboratory and clinical research for cancer gene therapy.Up to now,decoy ODN was adopted to inhibit STAT3 pathway in squamous cell carcinoma of the head and neck （SCCHN）,human breast cancer and human lung cancer.The results have showed that STAT3 signaling pathway could be significantly blocked,which subsequently resulted in the expression level downregulation of some oncogenes,including cyclinD1,c-myc, survivin,VEGF,bcl-2 and bcl-xl,and the depressed proliferation and increased apoptosis in the three cancer cells.It illuminates the study of therapeutic potential of STAT3 decoy ODN to human gliomas.In the current study,decoy ODN targeted inhibiting STAT3 pathway was employed to investigate the influence to human glioma cells.The study was divided into three parts.PartⅠOverexpression and Constitutive Activation of STAT3 in Human Glioma Samples and Cell LinesObjectiveTo study the expression level of STAT3 and its phosphorylation protein in human glioma cell lines and clinical specimens,and the significance of overexpression and constitutively activation of STAT3 signaling pathway in the pathogenesis of human glioma.Methods1.STAT3 and phosphorylated-STAT3 protein expression of 24 cases of human glioma samples were detected by Western blot analysis,and phosphorylated-STAT3’s frequence was evaluated in human glioma.2.The protein expression of human glioma cell lines U251 and A172 were detedted by Western blot analysis with anit-STAT3 and anti-phospho-specific STAT3 （Tyr705,Ser727）antibody.Results1.STAT3 were expressed at higher level in the human malignant glioma samples than normal neural tissues.Furthermore,in the human malignant glioma samples, 75%（18 of 24 cases）expressed Tyr-705-phospho-STAT3 and 66.67%（16 of 24 cases） expressed Ser-727-phospho-STAT3.In contrast,there was no present in nomal neural tissue.2.Overexpressed STAT3 and its two active forms,Tyr-705-phospho-STAT3 and Ser-727-phospho-STAT3,were identified in both U251 and A 172 cells.Conclusion1.Overexpression and constitutively activation of STAT3 are widely presented in human glioma samples and cell lines,it suggests that aberrant STAT3 signaling pathway may play an important role in the pathogenesis of human glioma.2.Overexpression and constitutively activation of STAT3 are presented in U251 and A172,so the two cell lines could be used as ideal models in our in vitro study.PartⅡTargeted Blockage of STAT3 Signaling Pathway with Decoy ODN Suppresses Growth of Human Glioma CellsObjectiveTo study the the transfection efficiency of STAT3 decoy ODN,the specificity of decoy ODN to STAT3 signaling pathway,and the inhibiting potency of STAT3 decoy ODN in glioma cell proliferation.Methods1.STAT3 decoy ODN was transfected into U251 and A172 cells as mediated by Lipofectamine^TM2000.2.Flow cytometry was used to examine the transfection efficiency of STAT3 decoy ODN in U251 andA172 cells.3.Fluorescence microscope was used to observe whether STAT3 decoy ODN was transfected into U251 and A172 cells.4.Luciferase reporter assay was used to examine the specificity of decoy ODN to STAT3 signaling pathway.5.Cell counting assay was used to examine the inhibitory effect of STAT3 decoy ODN in glioma cells.6.MTT assay was used to analyze the proliferation inhibiting effects in U251 and A 172.7.[³H]-thymidine incorporation assay was performed to detect the inhibiting consequence of two cell lines DNA synthesizing.Results1.STAT3 decoy ODN could be highly efficiently transfected into glioma cells as mediated by Lipofectamine^TM2000.The transfection efficiency detected with flow cytometry showed that the decoy ODN was introducted into U251 and A172 cells efficiently in a dose-dependent manner.The MFI means of U251 and A172 cells reached a maximum of 64.03%and 56.22%at 50 nmol/L decoy ODN,respectively. Moreover,according to the viewing by fluorescence microscope,the decoy ODN had been incorporated into glioma cells,partly in the nuclei.2.Decoy ODN specifically blocked STAT3 signaling pathway in glioma cells. According to STAT3-1uciferase reporter assay,STAT3 decoy ODN substantially reduced transcription activity of STAT3 in U251 and A172 cells（P＜0.05）.However,it didn’t cause similar decrease in STAT6 transcription activity.Additionally,by western blot analysis,we found that STAT3 decoy ODN did not have significant effect on the level of phosphorylated STAT3（Tyr705 and Ser727）in U251 and A172 cells.3.STAT3 decoy ODN could effectively inhibit cell proliferation in human glioma.The result of cell counting assay demonstrated that the growth of both U251 and A172 cells were inhibited significantly after treatment with STAT3 decoy ODN and exhibited a time- and dose-dependent manner（P＜0.05）.Consistent with the observation,the inhibition rate detected by MTT assay also confirmed that STAT3 decoy ODN contributed to potent growth inhibition in the two cell lines in a time-and dose-dependent manner（P＜0.05）.[³H]-TdR incorporation assay revealed that CPM gradually reduced for the increase of STAT3 decoy ODN.Compared with the control group（TE）,it was significant for DNA synthesis inhibition dued to 25 and 50 nmol/L STAT3 decoy ODN（P＜0.05）.Conclusion1.STAT3 decoy ODN can be highly efficiently transfected into glioma cells as mediated by Lipofectamine^TM2000.2.Decoy ODN can specifically block STAT3 signaling pathway by inhibiting its transcription activation.3.STAT3 decoy ODN can contribute to the growth suppression in human glioma cells with little side effect.PartⅢRelevant Mechanisms of Proliferative Inhibition via Targeted Blocking STAT3 Signaing Pathway with Decoy ODN in Human Glioma Cells ObjectiveTo study the relevant mechanisms on proliferative inhibition of targeted blocking STAT3 signaling pathway by the decoy ODN in the glioma cell lines.Methods1.Flow cytometry was used to examine the change of cell cycle after STAT3 decoy ODN were transfected into U251 and A172 cells.2.Annexin V/PI dual staining assay was used to examine whether cell apoptosis was induced after STAT3 decoy ODN were transfected into U251 and A172 cells.3.RT-PCR assay was used to examine the mRNA expression of STAT3-targeted genes after STAT3 decoy ODN were transfected into U251 and A172 cells.4.Western blot analysis was used to examine the protein expression of STAT3-targeted genes after STAT3 decoy ODN were transfected into U251 and A172 cells.Results1.STAT3 decoy ODN resulted in cell cycle arrest from G1 to S phase.Twentyfour hours after treated with 50 nmol/L STAT3 decoy ODN,U251 and A172 cells both exhibited a significant increase,cells in G0/G1 phase from 49.58±1.09%to 54.89±1.35%in U251（P＜0.05）,from 63.52±1.61%to 72.03±1.14%in A172 （P＜0.05）,and accompanied by a distinguished decrease in S phase,from 42.36±1.50%to 37.89±0.67%in U251（P＜0.05）,from 22.23±0.51%to 16.00±0.40%in A172（P＜0.05）.The cells treated with the mutant control decoy ODN showed no obvious alterations.2.STAT3 decoy ODN induced apoptosis in glioma cells.Annexin V/PI staining assay showed that treatment with STAT3 decoy ODN could lead to a significant increase in apoptosis,from 5.44±0.50%to 23.57±9.76%in U251 cells（P＜0.05）, and from 5.20±0.24%to 22.48±0.85%in A172 cells（P＜0.05）.In contrast,mutant control decoy ODN only induced 6.81%and 6.16%apoptosis respectively（P＞0.05）.3.STAT3 decoy ODN could down-regulate the expression of STAT3 targeted genes in glioma cells.As showed in RT-PCR,the mRNA levels of c-myc,cyclin D1, and bcl-xl in U251 cells were decreased by 52%,50%and 27.67%,respectively （P＜0.05）.In A172 cells,the mRNA levels of c-myc,cyclinD1,and bcl-xl decreased by 46.67%,40.67%,and 28.87%respectively（P＜0.05）.In contrast,the mutant control decoy ODN showed no effect on the mRNA levels of the malignant glioma cells（P＞0.05）.As shown in Western bolt analysis,compared with control,the protein expression levels of cyclinD1 and bcl-xl in U251 cell decreased 47.33%and 85.66%, respectively（P＜0.05）.The protein expression levels of cyclinD1 and bcl-xl in A172 cells were attenuated by 38.25%（P＜0.05）and 3.63%（P＞0.05）,respectively.Conclusion1.STAT3 decoy ODN result in cell cycle arrest from G1 to S phase in glioma cells.2.STAT3 decoy ODN can induce apoptosis in glioma cells.3.STAT3 decoy ODN can down-regulate the expression of STAT3 targeted genes in human glioma cells.STAT3 decoy ODN may symphysially contribute to human glioma cells growth suppression via the marked downregulation of the downstream oncogenes,and subsequent cell cycle arrest from G1 to S phase and cell apoptosis.In conclution, targeted blocking STAT3 signaling pathway with a decoy oligonucleotide may potentially be used as an effective anti-glioma therapeutic approach.更多还原