【作者】 马力；

【导师】 刘伟国；

【作者基本信息】 浙江大学 ， 外科学， 2010， 博士

【摘要】 [研究背景]胶质瘤是颅内最常见恶性肿瘤,其中病理分级为WHOⅢ-Ⅳ级的恶性胶质瘤,病情进展迅速,复发快,预后差。目前手术结合放、化疗的综合治疗是经典的治疗方法,但对于恶性胶质瘤患者的总体预后改善有限,主要原因在于难以避免的肿瘤复发。恶性胶质瘤的化疗是重要的辅助治疗手段,然而由于传统化疗药物的非特异性、细胞毒性和肿瘤本身的耐药性使得该类药物的总体疗效欠佳。近年来发现许多肿瘤的发生发展与特定信号通路的异常激活密切相关,因此以细胞特定受体、关键基因和调控分子为靶点的辅助治疗具有良好的应用前景。肿瘤干细胞假说认为在肿瘤细胞中仅有一小部分细胞具有自我更新、无限增殖、多向分化潜能的干细胞样特性,它们是肿瘤发生,转移和复发的根源。已经发现胶质瘤中也存在着这样一类以CD133阳性作为标记的脑肿瘤干细胞(BTSCs),并且证明其具有高度的耐药性、抗凋亡能力以及侵袭能力。这些肿瘤干细胞应该是今后恶性胶质瘤靶向治疗的主要研究方向。目前认为脑肿瘤干细胞可能起源于发生基因突变的正常神经干细胞,因而一些负责调控神经干细胞自我更新、分化、增殖的关键信号通路,如Hh、Wnt、Notch信号通路等,可能在脑肿瘤干细胞中起着重要的作用。其中Hh通路是目前肿瘤干细胞相关研究领域的热点之一。已有的研究表明：Hh通路既对内源性干细胞的增殖起调节作用,也可能对肿瘤干细胞起调节作用,其异常激活能导致多种肿瘤生成,如基底细胞癌、髓母细胞瘤、胶质瘤、胰腺癌等;目前体内外实验中对该通路的特异性阻断对这些肿瘤的治疗均取得了一定的效果,但其中的具体作用机制还没有完全阐明。因此对该信号通路的继续探索也成为了目前相关肿瘤基础研究领域的前沿。目前针对Hh通路应用较多的一类阻滞剂为环杷明(cyclopamine),其能够结合Smo受体从而抑制Hh信号通路活性。已有的体内外实验表明cyclopamine对包括部分胶质瘤在内的多种Hh通路相关性肿瘤有效。然而cyclopamine类抑制剂只能对由Hh信号通路上游基因变化导致异常活化的肿瘤有作用-而对由Smo级联反应以下的参与Hh信号通路调控的分子变化,如sufu基因突变所导致的肿瘤没有治疗效果。因此,进一步寻找Hh通路下游阻滞剂,如Gli转录因子阻滞剂,对于Hh通路相关性胶质瘤的干预治疗具有重要意义。[方法]第一部分：通过rt-PCR和免疫荧光技术检测Hh通路的主要基因Gli1在不同的原代细胞及胶质母细胞瘤细胞系中的表达；分别利用real-time PCR. western blot验证GANT61对Hh通路异常激活的胶质瘤Gli1 mRNA和Ptch1蛋白的调控作用；利用MTS试验,验证GANT61对不同株胶质瘤细胞活性的影响,并根据此项试验结果得出的时间-剂量-活性相关曲线确定该药物的有效浓度；进而借助于PI染色法检测GANT61对细胞周期的影响,并以Annexin V/PI双染色法检测细胞凋亡第二部分：利用免疫磁珠分选技术(MACS)在U87细胞系中分选得到CD133阳性的脑肿瘤干细胞,并通过单克隆形成试验、免疫荧光标记和裸鼠致瘤试验鉴定其干细胞样生物学特性;然后予以GANT61体外作用相应时间后再次检测脑肿瘤干细胞自我更新能力、干细胞特异性标记的维持以及裸鼠致瘤能力的变化。第三部分：通过裸鼠皮下接种法构建胶质瘤的种植瘤模型,按疗程瘤周注射GANT61后定期测量肿瘤体积变化;收集处理组与对照组新鲜肿瘤组织以PCR和免疫组化技术分别检测Ptchl mRNA和蛋白水平的变化；以TUNEL法标记凋亡细胞,以BRDU染色法检测肿瘤细胞的增殖。另取GANT61干预后的肿瘤组织体外再培养并检测其自我更新能力和体内致瘤能力。[结果]1.在7例恶性胶质瘤样本中3例为WHOⅢ级,4例为WHOⅣ级。其中2／5原代肿瘤细胞和1／2肿瘤细胞系的Gli1高表达。但Gli1的表达情况与病理分级没有明确的相关性。2.5μM浓度的GANT61能够在体外实验中时间依赖的阻断Gli1转录活性,3个样本作用48h后Gli1 mRNA均得到明显下调,作为Gli1下游靶基因之一的Ptch1的蛋白表达也较对照组下降,说明GANT61能通过抑制Gli1转录活性来阻断其下游的信号传导。3.GANT61对于Gli1表达异常增高的恶性胶质瘤具有时间和剂量依赖的细胞活性抑制作用,作用48h可以达到最大抑制效果,其IC50为10μM。4.10μM的GANT61作用48h可以明显增加G1亚峰和G0／G1期的细胞比例,这提示GANT61主要通过诱导G1／S期的周期阻滞和增加细胞死亡来减慢胶质瘤细胞增殖。5.10μM的GANT61作用48h可以通过降低Gli1转录活性而增加早期凋亡细胞比例。6.通过MACS技术从U87细胞系中分选出CD133阳性的细胞亚群。经验证分选所得的CD133阳性细胞具有自我更新、多向分化潜能和体内致瘤能力,具有脑肿瘤干细胞样细胞(BTSCs)的典型特征。7.CD133阳性组胶质瘤细胞Gli1 mRNA水平较阴性组明显增高。10μMGANT61作用48h后能明显抑制BTSCs Gli1转录活性。8.10μM GANT61共培养2天或20μM GANT61共培养7天都不能明显增加BTSCs早期凋亡,这提示在BTSCs中Hh通路可能并不是唯一调控其抗凋亡能力的信号通路。9.CD133阳性细胞与10μM GANT61共培养7天后,其肿瘤球样克隆形成能力明显减弱,CD133阳性率明显下降,将其接种至裸鼠皮下后未能致瘤。这提示GANT61对BTSCs的体外作用能降低其自我更新能力。10.用浓度5mg/ml、剂量20mg/kg的GANT61对皮下荷瘤裸鼠作隔天瘤周注射,2周后处理组种植瘤体积较对照组明显小11.对照组瘤体中Ptch1呈高表达,而在治疗组经过2周GANT61局部皮下注射后瘤体的Ptch1在mRNA和蛋白水平均明显下降。12.在体内环境下GANT61能提高肿瘤的TUNEL阳性率并降低BRDU标记的的阳性率,提示其能增加肿瘤细胞凋亡、抑制增殖。13.处理组种植瘤消化后经体外培养发现仍能形成肿瘤球样克隆,并具有裸鼠致瘤能力。这提示在体内实验中单独使用GANT61不能完全消灭肿瘤干细胞。[结论]1.在恶性胶质瘤中存在Hh通路活性异常增高的亚型,以Gli1 mRNA的高表达为主要表现。但Gli1的表达情况与病理分级没有明确的相关性。2.在体外实验中GANT61能通过抑制Gli1转录来降低胶质瘤细胞活性,这种作用主要通过G1／S周期阻滞和增加细胞凋亡来实现。3.体外分选得到的CD133阳性的BTSCs呈Gli1高表达,其能被GANT61所抑制,同时该药物还能抑制BTSCs自我更新能力,但对其抗凋亡能力无明显影响。4.体内实验中GANT61能通过抑制Gli1转录活性来抑制肿瘤细胞增殖、增加其凋亡,从而抑制肿瘤体积的增大。但该药物在体内未能完全根除BTSCs。更多还原

【Abstract】 [Background]Glioma is the most common malignancy in brain. Malignant glioma，which stands for WHO gradeⅢ-Ⅳ, is always associated with rapid progression, quick recurrence and poor prognosis. Currently radiotherapy and chemotherapy combined with surgical resection is the classic therapeutic strategy. However, due to inevitable relapse, the overall prognosis of patients with malignant glioma remains poor. Up to now，the effect of adjuvant chemotherapy is so limited, mainly because of the non-specificity, cytotoxicity of the chemical agents and the drug resistance of the tumor cells. Constitutive activation of some signal pathways has recently been implicated in the growth of a number of human malignancies. As a result, therapies which are targeting on such signal pathways, key genes and regulatory elements may have a good prospect. The cancer stem cell hypothesis implies that a small fraction of cells, called cancer stem cells (CSCs), present the stem cell ability such as indefinite proliferation, extensive self-renewal, and multi-linkage differentiation. They are the sources of tumor initiation, metastasis and relapse. The CD133 positive cells have been identified as brain tumor stem cells (BTSCs) in glioma. They display strong capability of drug resistance, anti-apoptosis, highly invasive thus represent novel targets for therapeutics. As BTSCs may originate from the transformation of normal neural stem cells (NSCs). similar signalling pathways, such as Hedrehog (Hh), Wnt and Notch pathways, may regulate self-renewal, proliferation and differenation in NSCs as well as in BTSCs. It has become clear that constitutive Hh signalling is an etiologic factor in many malignancies and application of specific Hh-pathway inhibitors has proven effective in preventing growth of many of these tumours in vitro and in vivo. However, the mechanism is not completely known. A detailed understanding of the role of Hh in the control of BTSCs behavior is critical for future therapeutic strategies.Current strategies focus on the use of small molecule inhibitors of Smo, such as cyclopamine et al. However, the approach is limited because only tumors where pathway activation has occurred at the level of Smo or upstream can be treated. Tumors with inactivating mutations in SuFu or other downstream components will not respond to these antagonists. In that case, downstream Hh pathway blocker is needed and antagonists against GLI transcription factors would be a better choice.[Method]Part 1:To identify whether Hh signaling pathway is active in glioma cell line or primary glioma cells. Rt-PCR and cytoimmunofluorescence were used to detect the Gli1 level in all of the samples. Real-time PCR and western blot were used to determine the influence of a GLI transcription antagonist GANT61 on Glil mRNA and Ptchl protein respectively. Then in order to determine whether GANT61 could inhibit glioma cell viability in a Gli1 level dependent way. MTS trials were performed in all 7 samples. Finally, cell cycle test and Annexin V/PI double staining were used to explore the mechanism leading to cell viability inhibition.Part 2:CD133 positive cells were isolated through MACS. These cells were verified as BTSCs using Single-cell clonal culture, cytoimmunofluorescence staining and xenograft tumor formation in nude mice. The Glil mRNA level was evaluated in both CD133 positive and CD133 negative subgroup using real-time PCR. After being cultured with GANT61 the Gli1 mRNA level. CD133 positive rate, the capacity of self-renewal and in vivo tumorigenecity were re-determinated to find out whether BTSCs can be depleted in vitro.Part 3:in order to evaluate the effect of GANT61 against glioma in vivo the drug was injected subcutaneously every other day for 2 weeks after establishment of xenograft tumors. Tumor volumes were calculated and BRDU as well as TUNEL staining were performed on the tissue section after remove of tumor. Finally, secondary tumorsphere formation ability and in vivo tumorigenecity were determined to evaluate whether all of the BTSCs have been depleded after treatment in vivo.[Results:]Seven samples were included in the experiment with 3 belonging to WHO gradeⅢand 4 belonging to WHO gradeⅣ. The Gli1 level was highly expressed in 2 of the primary glioma cells and 1 of the glioma cell lines. However. Gli1 level was not correlated with tumor grade.The Glil mRNA level and Ptchl protein level of the glioma cells were significantly decreased 48h after 5μM GANT61 administration. It suggested that Gli-mediated transcription in glioma cells was inhibited by GANT61 in vitro.Treatment of GANT61 inhibited glioma cell viability in a time and dose dependent way. The IC50 was about 10μM and the maxmal effect exhibited 48h after administration. The proportion of cells in sub-G1 and G0/G1 phase both increased significantly 48 h after 10μM GANT61 administration. It implied the agent suppressed glioma cell proliferation by means of cell-cycle arrest as well as increasing cell death.The proportion of early stage apoptosis increased significantly 48 h after 10μM GANT61 administration.CD133 positive cells were isolated through MACS. These cells were characterized as BTSCs because they hold the capacity of self-renewal. multi-linkage differentiation and in vivo tumorigenecity.The Gli1 mRNA level of the CD133 positive group was significantly higher than that of the CD133 negtive group. Treatment of 10μM GANT61 for 48h can significantly suppress the Gli1 mRNA.Neither 10μM GANT61 for 2 days nor 20μM GANT61 for 7 days could increase the proportion of early stage apoptosis in CD133 positive cells. It implies that inhibit Hh pathway only may not be enough to deprive the high capacity of anti-apoptosis in BTSCs.After blocking of Hh signaling for 7 days in vitro the CD133 positive rate as well as capacity of Single-cell tumorsphere formation and in vivo tumorigenecity of BTSCs dropped dramaticly. It implied that the self-renewal capacity of BTSCs may have been depleted by inhibition of Gli1 transcription in vitro.Treatment with GANT61 resulted in the inhibition of xenograft tumor growth and limited the increase in tumor size.The Ptchl mRNA level and protein level of the xenograft tumor were significantly suppressed after GANT61 administration. It suggested that Gli1-mediated transcription in glioma cells was inhibited by GANT61 in vivo.The TUNEL staining positive rate in GANT61-treated samples were significantly higher than that of the control while the BRDU positive rate was much lower. This result might indicate that GLI transcription inhibition favors induction of apoptosis and suppress tumor proliferation in vivo.Secondary tumorspheres originating from xenograft tumor of the treatment group can be found in culture favoring stem cell. It implies that despite the tumor shrinking some BTSCs can still survive after GANT61 administration in vivo.Conclusion1. Hh pathway was activated in a subset of malignant glioma including primary glioma samples and glioma cell lines. However,Gli1 level was not correlated with tumor grade. 2. Treatment of GANT61 inhibited glioma cell viability in a time and dose dependent way. This agent suppressed glioma cell proliferation by means of G1/S cell-cycle arrest as well as increasing cell apoptosis in vitro.3. Hh pathway activity was much higher in BTSCs than in CD133 negtive glioma cells. The self-renewal capacity and in vivo tumorigenecity but not anti-apoptosis capacity could be abolished by treatment of GANT61 in vitro.4. Subcutaneously administration of GANT61 could decrease glioma cell proliferation and increase apoptosis of xenograft tumor in nude mice. However, some BTSCs can still survive after treatment.更多还原