【作者】 申杰；

【导师】 李刚；

【作者基本信息】 山东大学 ， 神经外科， 2009， 博士

【摘要】 人脑胶质瘤是中枢系统最常见的原发性肿瘤,其中胶质母细胞瘤占颅内肿瘤的近50%。虽然近年来手术及术后的放疗和化疗取得了很大的进步,但不幸的是,其确诊后的生存期仍然让人失望,只有12-15个月。因此,新的治疗胶质母细胞瘤的方法在改善预后和病人生活质量方面极需提高。到目前为止,人们普遍认为肿瘤的发生与发展是一个多种基因参与、多环节、多阶段、多步骤的复杂过程。所以仅仅干预一个肿瘤相关基因很难达到治疗胶质瘤的目的。近年来,一些转录因子被逐渐发现,它们在调节多个肿瘤相关基因方面起重要作用。换句话说,这些转录因子可以看作是肿瘤发生和发展的总开关。因此,靶向性阻断某个转录因子治疗肿瘤越来越受到人们的关注,也逐渐成为治疗胶质瘤的一个新策略。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,存在于胞浆,参与调节胚胎发育,细胞的正常生长、分化和恶变等过程。持续活化的STAT3能够调控许多肿瘤发生的环节,包括细胞周期,凋亡,肿瘤血管形成及肿瘤细胞逃逸免疫系统等。因此它被看作癌基因并且实验证实阻断STAT3通路后可以使肿瘤细胞的增殖受到抑制,凋亡增加。这些都为STAT3作为分子靶点治疗胶质瘤提供了强有力的证据。到目前为止,已经有几种阻断STAT3通路的策略,包括显性负性调节,反义核苷酸,RNA干扰技术,生化制剂,双链或单链诱骗寡核苷酸技术。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。由于它的高效,低成本和高特异性等优点,诱骗寡核苷酸技术广泛地应用于包括乳腺癌、肺癌、胰腺癌和头颈部鳞癌在内的多种肿瘤的研究。结果显示,阻断STAT3通路后肿瘤细胞的生长显著受到抑制并引起凋亡,然而,对胶质瘤在体内生长情况的影响却鲜为人知。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体内效应和机制。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基础。方法1、Western印迹方法检测60例人脑胶质瘤标本和5例正常脑组织标本中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统计学分析。2、Western印迹方法检测人脑胶质瘤细胞系U251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及其磷酸化的异常表达。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80%（60例中有48例）,丝氨酸磷酸化的表达阳性率为60%（60例中有36例）。而在正常脑组织中两种磷酸化均未明显表达。2、人脑胶质瘤细胞系U251中存在STAT3及其磷酸化（705位酪氨酸磷酸化STAT3与727位丝氨酸磷酸化STAT3）的异常表达。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基础。2、U251细胞系中存在STAT3及其磷酸化的异常表达,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并促进其凋亡的体外研究目的研究阳离子脂质体转染STAT3 Decoy-ODN的效果和效率。检测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。检测Decoy-ODN转染人脑胶质瘤细胞对其凋亡的影响。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内。2、流式细胞术检测FITC标记的STAT3 Decoy-ODN在U251中的转染效率。3、细胞计数法检测Decoy-ODN靶向性阻断STAT3信号通路后对人脑胶质瘤细胞增殖的抑制效应。4、Annexin V/PI双染法检测Decoy-ODN靶向性阻断STAT3信号通路后U251细胞的凋亡情况。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高效转染入人脑胶质瘤细胞。流式细胞术检测结果显示,100 nmol/L FITC-STAT3 Decoy-ODN被Lipofectamine ^TM2000介导转染后,U251细胞的转染效率是88.71%。2、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能够抑制胶质瘤增殖,抑制效应和时间及ODN浓度呈正相关。3、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,25 nmol/L STAT3 Decoy-ODN干预24小时后,U251细胞凋亡率从1.23±0.48%增长至39.54±5.62%（P＜0.05）,作为对照,Scramble-ODN诱导的U251凋亡细胞仅增加了大约4.14%,无统计学意义（P＞0.05）。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高效转染入人脑胶质瘤细胞内。2、STAT3 Decoy-ODN在体外能够抑制人脑胶质瘤细胞增殖。3、STAT3 Decoy-ODN在体外能够促进人脑胶质瘤细胞凋亡。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体内研究目的评价裸鼠皮下种植U251细胞的效果,评价瘤内注射Decoy-ODN的效果,检测在体内Decoy-ODN对人脑胶质瘤细胞增殖和凋亡的影响,并对其相关基因和蛋白的表达水平进行统计学分析,进而深入探讨在体内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、皮下注射法将U251细胞注入4周龄裸鼠右胁腹皮下。2、观察约一周时间,皮下瘤结节能明显触到后,瘤内注射Decoy-ODN并检测其在体内对人脑胶质瘤细胞生长的影响。3、免疫组织化学法检测体内STAT3 Decoy-ODN作用人脑胶质瘤细胞后对增殖标志物Ki67表达水平的影响。4、Tunel法检测在体内Decoy-ODN对人脑胶质瘤细胞凋亡的影响。5、RT-PCR检测分析在体内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。6、免疫组织化学法检测体内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、U251细胞可以在裸鼠体内成瘤。成瘤后随机分3组,进行瘤内注射。与对照组相比,40天后经PBS处理组的肿瘤平均体积为884±111 mm³,而Decoy-ODN注射组的肿瘤平均体积为265±33 mm³,体积缩小70%（P＜0.05）,Scramble-ODN注射组肿瘤平均体积为713±90 mm³,体积缩小20%（P＞0.05）,无统计学意义。2、STAT3 Decoy-ODN能够在体内抑制胶质瘤细胞的增殖。免疫组织化学法检测切片中增殖标志物Ki67的表达情况,发现STAT3 Decoy-ODN组中Ki67的表达量与对照组相比减少73.2%,有显著性差异（P＜0.05）。3、STAT3 Decoy-ODN能够在体内诱导胶质瘤细胞凋亡。Tunel法检测结果表明,与对照组相比,Decoy-ODN组中每高倍视野中阳性细胞数48.3±9个,Scramble-ODN组中每高倍视野中阳性细胞数9.3±1.5个,分别是对照组的5.6倍和1.1倍（P＜0.05）。4、STAT3 Decoy-ODN能够在体内下调胶质瘤中细胞周期和抗凋亡相关基因的表达水平。RT-PCR检测结果显示,Decoy-ODN组的c-myc、cyclinE、cyclinD1、bcl-xl、bcl-2和survivin的mRNA表达水平分别下调了53.3%、46.3%、64.5%、50.7%、58.6%和69.6%,和对照组比较,有显著性差异（P＜0.05）。5、STAT3 Decoy-ODN能够在体内降低细胞周期和抗凋亡相关蛋白的表达。切片的免疫组织化学结果显示,Decoy-ODN组的cyclinD1、bcl-xl和bcl-2的蛋白表达水平分别下调了68.5%、68.8%和75.1%。和对照组比较,有统计学意义（P＜0.05）。结论1、U251细胞能够在裸鼠体内成瘤。2、STAT3 Decoy-ODN在体内能够显著抑制人脑胶质瘤细胞生长。3、STAT3 Decoy-ODN在体内能够显著抑制人脑胶质瘤细胞增殖。4、STAT3 Decoy-ODN在体内能够显著诱导人脑胶质瘤细胞凋亡。5、STAT3 Decoy-ODN在体内能够下调细胞周期和抗凋亡相关基因的表达。6、STAT3 Decoy-ODN在体内能够降低细胞周期和抗凋亡相关蛋白的表达。总之,通过本研究表明,在体内应用STAT3 Decoy-ODN能够抑制人脑胶质瘤的生长,增殖并诱导细胞凋亡。下调胶质瘤细胞中细胞周期和抗凋亡相关基因的表达,同时降低细胞周期和抗凋亡相关蛋白的表达。为人脑胶质瘤的体内基因治疗提供了一条有效途径。更多还原

【Abstract】 Human malignant gliomas are the most common type of primary brain tumors, and glioblastoma multiforme （GBM） accounts for more than 50% of all intracranial gliomas. Regardless of advances in surgical resection, followed by radiotherapy and chemotherapy, unfortunately, the median survival time remains discouraging at 12-15 months from diagnosis. Novel therapies are needed to improve the prognosis and quality of life of patients with GBM. So far, it’s well known that Carcinogenesis is a multiple-link, multi-stage, and multi-step process, which involves in a great number of oncogenes and antioncogenes. Therefore, it is difficult to treat glioma only by intervening one cancer-related gene. In recent years, some transcription factors have been discovered, which plays an important role in modulating multiple cancer-related genes. In other words, the transcription factors can be considered as a general switch of cancergenesis and aggravation. Therefore it has drawn more and more attentions on targeted inhibiting a specific transcription factor to treat variety of cancers, including human glioma and others.Signal transducer and activator of transcription 3 （STAT3）, a member of the Janus kinase （JAK）/STAT signaling pathway, is a central cytoplasmic transcription factor, that is involved in the embyro development, physiologic regulation of cell growth, survival and differentiation, and pathogenesis of malignancies. The constitutive activation of STAT3 regulates a number of pathways important in tumorigenesis including cell cycle progression, apoptosis, tumor angiogenesis, and tumor cell evasion of the immune system. So STAT3 has been considered as an oncogene and many experiments have identified that blockade of STAT3 signaling pathway could result in a depressed proliferation and increased apoptosis in human cancer cells. The results offer strong evidences that STAT3 acts as a promising molecular target of gene therapy for glioma.Until now, several strategies were performed to block the signaling pathway of STAT3, including dominant negative, antisense, interference （siRNA）, biochemical agent, double-stranded or single-stranded "decoy" oligonucleotides. The Decoy-ODN strategy is based on the competition 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 cis-elements; prevent the transcription and activation of the oncogene, and subsequent result in cells apoptosis and inhibition of glioma cells proliferation. Due to its high efficiency, low cost and high specificity, Decoy-ODN strategy has been adopted in many cancers, such as breast cancer, lung cancer, pancreas cancer, and squamous cell carcinoma of the head and neck （SCCHN）. The results have showed that after STAT3 signaling pathway was significantly blocked, the cancer cells were significantly suppressed growth and induced apoptosis. But the condition in glioma in vivo is known little. In the present study, the Decoy-ODN targeted inhibiting STAT3 pathway was employed to investigate the influence on human glioma cells in vivo. The study was divided into three parts.Part I Overexpression and Constitutively Activation of STAT3in Human Glioma Samples and U251 Cell LineObjectiveTo 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 60 cases of human glioma samples and 5 cases of human normal brain tissue were detected by Western blot analysis, and phosphorylated-STAT3’s frequence was evaluated in human glioma.2. The protein expression of human glioma cell line U251 was detected by Western blot analysis with anit-STAT3 and anti-phospho-specific STAT3 （Tyr705, Ser727） antibody.Results1. STAT3 existed in the human malignant glioma samples and normal brain tissues. Furthermore, in the human malignant glioma samples, 80% （48 of 60 cases） expressed Tyr-705-phospho-STAT3 and 60% （36 of 60 cases） expressed Ser-727-phospho-STAT3. In contrast, there was no obvious expression of phospho-STAT3 in nomal brain tissue.2. Overexpressed STAT3 and its two active forms, Tyr-705-phospho-STAT3 and Ser-727-phospho-STAT3, were identified in U251 cells.Conclusion1. Overexpression and constitutively activation of STAT3 is widely present in human glioma samples, suggesting that aberrant STAT3 signaling pathway play an important role in the pathogenesis of human glioma.2. Overexpression and constitutively activation of STAT3 is present in U251 cell line, that is, it could be used as an ideal model in vivo.Part II Targeted Blocking STAT3 Signaling Pathway with Decoy-ODN Suppresses the Growth and Induces the Apoptosis of Human Glioma Cells invitroObjective To study the transfection efficiency of STAT3 Decoy-ODN in U251 cells. To assay the inhibiting potency of STAT3 Decoy-ODN in glioma cell proliferation and the influence on apoptosis of glioma cells.Methods1. STAT3 Decoy-ODN was transfected into U251 cells mediated by Lipofectamine^TM2000.2. Flow cytometry was used to examine the transfection efficiency of STAT3 Decoy-ODN in U251 cells.3. Cell counting assay was used to examine the inhibitory effect of STAT3 Decoy-ODN in glioma cells.4. Annexin V/PI dual staining assay was used to examine cell apoptosis after transfection of STAT3 Decoy-ODN into U251 cells.Results1. STAT3 Decoy-ODN could be highly efficiently transfected into glioma cells by Lipofectamine^TM2000. The transfection efficiency detected by flow cytometry showed that the Decoy-ODN was efficiently transfected into U251 in a dose-dependent manner. The transfection efficiency of U251 cells reached a maximum of 88.71% at 100nmol/L Decoy-ODN.2. STAT3 Decoy-ODN could effectively inhibit cell proliferation in human glioma. The result of cell counting assay demonstrated that the growth of U251 cells were inhibited significantly after treatment with STAT3 Decoy-ODN and exhibited in a time- and dose-dependent manner （P<0.05）.3. 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 1.23±0.48% to 39.54±5.62% in U251 cells. In contrast, Scramble-ODN only induced apoptosis at 4.14% （P>0.05）. Conclusion1. STAT3 Decoy-ODN can be highly efficiently transfected into glioma cells mediated by Lipofectamine?2000.2. STAT3 Decoy-ODN can suppress the growth of human glioma cells with little side effect.3. STAT3 Decoy-ODN can induce apoptosis of human glioma cells with little side effect.Part III Targeted Blockade of STAT3 Signaling Pathway with Decoy-ODN Has Inhibitory Effect on Human Malignant Glioma Cells in vivoObjectiveTo evaluate the effect of subcutaneous xenografts of U251 cells in nude mice. After intratumoral injection of STAT3 Decoy-ODN, the expression of caner-related genes and proteins in vivo are analyzed. Further, to study the relevant mechanisms on proliferative inhibition of targeted blocking STAT3 signaling pathway by the Decoy-ODN in the glioma cell lines.Methods1. U251 cells were subcutaneously injected into the right flank of 4-weeks old nude mice.2. Decoy-ODN was intratumorally injected after the tumor formation. The volume of the tumor was measured to evaluate the suppression of tumor growth.3. Ki67 was considered as the marker of proliferation and measured by immunohistochemistry.4. The effect of apoptosis in vivo was measured by Tunel.5. RT-PCR assay was used to examine the mRNA expression of STAT3 targeted genes in vivo.6. Immunohistochemistry analysis was used to examine the protein expression of STAT3- targeted genes in vivo.Results1. U251 cells could form xenografts in the nude mice. STAT3 Decoy-ODN was directly delivered into xenografts after the nude mice randomly divided into 3 groups. Contrast to control group with 884±111 mm³ in average volume, the average volume of xenografts in Decoy-ODN treated group was 265±33 mm³ , decreased by 70%（P<0.05）, while in Scramble-ODN treated group was 713±90 mm³, decreased by 20% （P>0.05）.2. STAT3 Decoy-ODN suppressed proliferation of glioma cells in vivo. Ki67 labeling index decreased by 73.2% after treatment with decoy-ODN relative to that observed in the PBS or scramble-ODN groups （P<0.05）.3. STAT3 Decoy-ODN induced apoptosis in glioma cells in vivo. Tunel analysis showed that 48.3±9 positive cells in per high power field in the group treatment with STAT3 Decoy-ODN, while in Scramble-ODN group, only 9.3±1.5 positive cells, that is, as 5.6-fold and 1.1-fold as control group, respectively （P<0.05）.4. STAT3 Decoy-ODN could down-regulate the expression of STAT3 targeted genes in glioma cells in vivo. As showed in RT-PCR, the mRNA levels of c-my、cyclinE、cyclinD1、bcl-xl、bcl-2 and survivin in Decoy-ODN treatment group were decreased by 53.3 %、46.3%、64.5%、50.7%、58.6% and 69.6%, respectively （P<0.05）. In contrast, the Scramble-ODN showed no effect on the mRNA levels of the malignant glioma cells in vivo.5. STAT3 Decoy-ODN could down-regulate the protein expression of STAT3 targeting genes in glioma cells in vivo. As shown in immunohistochemistry analysis, compared with control, the protein expression levels of cyclinD1、bcl-xl and bcl-2 in STAT3 Decoy-ODN treatment group decreased by 68.5%、68.8% and 75.1%, respectively （P<0.05）.Conclusion1. U251 cells can form xenografts in the nude mice.2. STAT3 Decoy-ODN can suppress growth of glioma cells in vivo.3. STAT3 Decoy-ODN can inhibit proliferation of glioma cells in vivo.4. STAT3 Decoy-ODN can induce apoptosis of glioma cells in vivo.5. STAT3 Decoy-ODN can down-regulate the gene expression of STAT3 targeted genes in human glioma cells in vivo.6. STAT3 Decoy-ODN can down-regulate the protein expression of STAT3 targeting genes in human glioma cells in vivo.In conclusion, STAT3 Decoy-ODN could suppress growth, inhibit proliferation and induce apoptosis of human glioma cells via the significant downregulation of the downstream oncogenes and the low expression of related proteins in vivo. Furthermore, it is considered as a potentially effective gene therapeutic approach for glioma in vivo.更多还原