【作者】 范海涛；

【导师】 庞琦；

【作者基本信息】 山东大学 ， 外科学， 2012， 博士

【摘要】 研究背景和目的恶性胶质瘤是最常见的中枢神经系统肿瘤之,并具有极高的死亡率。即使经过积极的治疗,其中位生存时间仍不乐观。胶质母细胞瘤生存时间约12-15个月,间变性星形细胞瘤(WHO Ⅲ)约2-3年,弥漫性星形细胞瘤(WHOll)的也仅5-10年。在脑肿瘤内有一群具有肿瘤干细胞(TSC)特点的细胞,能够表达神经干细胞(NSC)标记物CD133和巢(nest.in)蛋白。这些所谓的“胶质瘤干细胞”具有无限增殖、侵袭等特点,因此对肿瘤的起源、生长及复发有着可不推卸的责任。尽管目前对胶质瘤干细胞仍存有很多争议,但不妨碍其对生物医学界与日俱增的吸引力。肿瘤干细胞的许多分子及功能特点,将来有可能被应用于发展新型抗肿瘤药物中。随着对胶质瘤增殖、侵袭及血管形成的病理及分子机制研究取得的巨大进步,基因治疗作为一种高效的治疗手段得到迅速发展,然而选择恰当的肿瘤基因作为目标靶基因非常的困难。高迁移率族蛋白A1(HMGA1)是一种非组蛋白、核结构性转录因子,其编码基因位于染色体6p21.3。HMGA1含有3个被称为“AT-hook"的DNA绑定区域,其能使HMGA1在染色体的小沟处与富含AT碱基的DNA序列相结合。虽然HMGA1本身并没有转录活性,但是其可以通过蛋白与蛋白、蛋白与DNA的相互作用来改变染色体的结构,进而调节转录过程。HMGAl参与各种生物学过程,其中包括基因转录、胚胎形成、细胞周期的调节、凋亡、肿瘤形成。尽管HMGA1在胚胎时期广泛的表达,但是在成熟分化的人体组织包括脑组织中表达极低甚至没有。近来的研究发现HMGA1在人体良、恶性肿瘤中广泛的表达,然而其在肿瘤产生的过程中所起的作用一直没有得到诠释。除胶质瘤外,大脑另一类主要的恶性肿瘤是颅内转移瘤。大脑是肿瘤转移的常见部位,全身其它部位肿瘤约1/3将最终发生中枢神经系统(CNS)转移。这主要是由于血脑屏障及大脑的“免疫特权”地位,造成化疗药物及炎症细胞不能够进入中枢神经系统的结果。Toil样受体(TLR)家族作为一类模式识别受体,在脑巨噬细胞(MP)及小胶质细胞(MG)中广泛的表达。TLR的激活能够增加炎症细胞的吞噬作用,促进Thl细胞因子的分泌,调节趋化白细胞聚集到感染组织。动物实验证实,CpG寡聚脱氧核苷酸(CpG)这类TLR激活剂可以作为肿瘤疫苗免疫辅助剂与TLR9结合,诱导产生适应性、抗原特异性的细胞抗肿瘤免疫反应。然而对胶质瘤及黑色素瘤病人早期的临床跟踪回访发现,其结果不是非常的乐观。尽管大剂量的CpG能够有一定的临床效果,但是由于CpG引起严重的炎症反应致使大脑水肿,而使这一抗肿瘤作用受到限制。近来我们的研究发现小剂量的CpG与碳纳米管(CNT)结合不但能够治愈胶质瘤,而且产生的系统抗肿瘤免疫反应能预防肿瘤的复发。本论文共分三部分：1.从胶质瘤细胞株U251中提取胶质瘤干细胞,研究HMGA1在胶质瘤细胞株U251及提取的干细胞中的表达差异,进而从胶质瘤干细胞的角度证明HMGA1的表达与肿瘤的恶性度有关。2.探讨HMGA1在不同WHO分级的星形胶质瘤中的表达差异及与肿瘤增殖、侵袭、血管形成能力的关系。3.利用有生物学功能的碳纳米管与CpG结合增加CpG的抗肿瘤活性,并用载瘤小鼠动物模型予以验证。我们提出假设并予以证明,颅内应用CNT-CpG复合体的抗肿瘤治疗,其效果可能超过一般的系统免疫治疗,它产生免疫抗肿瘤反应不但能够抑制颅内转移瘤的生长,而且还能抑制原发部位肿瘤的生长。第一部分HMGA1在胶质瘤细胞株U251和U251提取的胶质瘤干细胞中的表达差异目的：从胶质瘤细胞株U251中提取胶质瘤干细胞(GSC)。探讨HMGA1在胶质瘤细胞株U251及提取的干细胞中的表达,进一步从肿瘤干细胞(TSC)的角度证明HMGA1的表达与肿瘤的恶性度有关。方法：利用免疫磁珠从胶质瘤细胞株U251中提取表面表达CD133的GSC,并利用流式细胞计数、免疫荧光证明所提取的细胞为GSC。利用RT-PCR和Western Blotting检测HMGA1在U251及提取的干细胞中的基因与蛋白表达差异。结果：1.从胶质瘤细胞株U251中成功提取了GSC,并用无血清培养基进行培养。胶质瘤干细胞在U251中所占的比例约为0.32±0.07%。2.HMGA1在GSC中的表达远高于普通胶质瘤细胞(P<0.05),其中在基因及蛋白水平的差距分别为6.13±0.25倍、2.75±0.99倍。结论：实验结果显示HMGA1在提取的GSC中的表达远高于原U251细胞。从TSC的观点证实HMGA1的表达与肿瘤增殖、侵袭及肿瘤分化程度有关系。因此HMGA1可以作为一个潜在的靶向治疗指标,用于胶质瘤及干细胞的治疗研究中。第二部分HMGA1在恶性胶质瘤中的表达及与肿瘤的增殖、侵袭、血管形成能力的关系目的：研究HMGA1在人恶性胶质瘤不同WHO分型中的表达差异,并证明HMGA1的表达与肿瘤的增殖、侵袭、血管形成能力相关。方法：选取64例山东大学附属省立医院神经外科收治患者的手术标本；其中包括来自脑挫裂伤病人的正常脑组织4例,弥漫性星形细胞瘤22例,间变性星形细胞瘤23例,胶质母细胞瘤15例。利用免疫组织化学和实时定量RT-PCR测量HMGA1在恶性胶质瘤不同WHO分型中的表达差异,及与恶性肿瘤增殖、侵袭、血管形成能力的关系。结果：1.免疫组织化学结果显示：HMGA1蛋白为细胞核染色蛋白,在正常脑组织中表达为阴性；恶性胶质瘤中的表达为96.7%(58例/60例),其中包括强阳性表达(+++)的15例(25.0%),中度表达(++)的28例(46.7%),阴性或弱阳性(0-+)的17例(28.3%)。HMGA1在胶质母细胞瘤中的表达,远高于WHOⅡ(P=0.002)和WHOⅢ(P=0.024)星形细胞瘤。HMGA1蛋白的表达与Ki-67(r=0.53,P=0.00)、MMP-9(r-0.508,P=0.000)、VEGF-A (r=0.316,P=0.014)和血管密度MVD (r=0.321,P=0.012)密切相关。2.实时定量PCR的结果也支持上述观点。基因水平上,HMGA1在胶质母细胞瘤的表达也高于WHO Ⅱ(P=0.043)和WHOⅢ(P=0.031)星形细胞瘤；HMGA1的表达与Ki-67(r=0.429,P=0.025). MMP-9(r=0.443. P=0.024)、VEGF-A (r=0.409, P=0.034)密切相关。结论：实验结果显示HMGA1在人恶性胶质瘤中的表达与胶质瘤的恶性度、增殖能力、侵袭性、血管形成能力密切相关。因此HMGA1可以作为一个生物学标记用于人类肿瘤的诊断与治疗中。第三部分CpG联合碳纳米管颅内免疫抑制黑色素瘤原发灶及颅内转移灶的生长目的：有生物学功能的碳纳米管(CNT)与CpG结合,增加CpG的免疫抗肿瘤活性,并用载瘤小鼠模型予以验证。本实验我们提出了假设并给予证明：颅内应用CNT-CpG优于其它的系统抗肿瘤治疗,其能够激起全身的免疫抗肿瘤活性,能同时抑制黑色素瘤原发灶和颅内转移灶的肿瘤生长。方法：体外实验,利用Raw巨噬细胞分泌转录因子蛋白NF-κB来测量TLR-9的活性。体内实验,利用Xenogen IVIS成像系统来测量CpG及CNT-CpG的生物学分布；Cobra量子伽马计数仪分析细胞毒性；流式细胞计数和FlowJo8.4.7分析软件来分析测量不同类型的细胞,并计算炎症细胞所占的比例。结果：1.体外实验显示,CNT-CpG应用24小时后,激活NF-κB的能力是单纯应用CpG的2倍以上2.休内实验显示,无论是CNT-CpG-Cy5.5还是单纯的CpG-Cy5.5,其在颅内黑色素瘤中的代谢时间要比原发部位肿瘤长。甚至注射7天后,颅内Cy5.5荧光信号仍能够被发现。同时我们对比CNT-CpG和单纯的CpG在颅内黑色素瘤中的生物学分布结果发现,单纯的CpG注射后,从注射部位向远处弥散；然而CNT-CpG却一直聚集在肿瘤及其边缘。3.相对于单纯小剂量应用CpG或CNT, CNT-CpG不但能够引起局部的抗肿瘤反应,还能够激活全身的抗肿瘤反应。颅内应用CNT-CpG虽然能够延长载瘤小鼠的生存周期,但是不能像治愈胶质瘤那样治愈黑色素瘤的颅内转移灶及原发灶。4.颅内应用CNT-CpG能够增加MP(CD45hiCD11b+)、NK、CD8及CD4向颅内肿瘤的迁移,但不能够使上述细胞向非治疗的原发部位迁移。但是相对于原发部位治疗,颅内应用CNT-CpG的小鼠所取的脾细胞有很强的体外抗肿瘤能力。结论：我们利用小鼠黑色素瘤模型证明了纳米颗粒在优化CpG免疫治疗方面的高效性。更有意义的是,证实了颅内应用CNT-CpG复合体治疗颅内转移瘤的潜在价值。颅内应用CNT-CpG后,肿瘤相关小胶质细胞能更够高效的摄取CNT-CpG,并聚集在肿瘤及周围,产生强烈的免疫抗肿瘤反应。CNT-CpG复合体不但能够产生局部的抗肿瘤反应,还能够产生系统的全身抗肿瘤免疫反应。更多还原

【Abstract】 Background and ObjectiveMalignant glioma is the most common central nervous system (CNS) tumor and carries an extremely high mortality rate. Despite optimal treatment, the median lengths of survival is only12to15months for patients with glioblastomas multiforme (GB), is2to3years for patients with anaplastic astrocytoma (AA, WHO grade III) and5to10years for patients with diffuse astrocytoma (DA, WHO grade II). A small subpopulation of cells with tumor stem cells’(TSCs’) properties has been identified in brain tumors that express the neural stem cell (NSC) markers CD133and nestin protein. These cells have many distinct characteristics of stem cells including limitless proliferation, invasion, and were responsible for the genesis, growth and recurrence of tumor. Although there are some different voices about this hypothesis, it still draws more and more interests. The molecular and functional characterization of cancer stem cell may be used in the development of novel cancer therapeutic drugs.With the dramatic progress made in pathogenesis and molecular mechanisms of proliferation, invasion and angiogenesis about malignant gliomas, gene therapy is being developed as a more effective strategy in treatment, but it is very difficult to select proper tumor genes for targeted therapies. The high-mobility group Al (HMGA1) protein is a non-histone architectural nuclear factor and encoded by the gene at chromosomal loci6p21.3. HMGA1contains three basic DNA-binding domains termed "AT-hooks", which mediate binding to the AT-rich regions in the narrow minor groove of DNA. Although HMGA1has no intrinsic transcriptional activity alone, through protein-protein and protein-DNA interactions, it can modulate transcription by altering chromatin architecture. It participates in diverse biological processes, including gene transcription, embryogenesis, cell cycle regulation, apoptosis and even neoplastic transformation. Although widely expressed during embryonic development, its expression level is negligible or absent in fully differentiated adult tissues including normal brain tissues. Recently published studies showed that HMGAl was overexpressed in several human benign and malignant tumors. However, the role of HMGA1in tumor progression is still not clear.Besides glioma, another most common type of brain tumor is brain metastasis. The brain is a common location for tumor metastasis; approximately one third of patients with systemic cancer ultimately develop central nervous system (CNS) involvement. This is due in part to the blood-brain barrier and the brain’s "immune-privileged" status that each contribute to prevent chemotherapeutic drugs and inflammatory cells from penetrating into the CNS. Toll-like receptor (TLR)-family members as pattern-recognition receptors are wildly expression in brain microglia (MG) and macrophages (MP). Activation of toll-like receptors (TLRs) has been shown to enhance phagocytosis, promote secretion of Thl cytokines, and mediate leukocyte recruitment to infected tissues. Consequently, agonists such as CpG oligodeoxynucleotides (CpG) that bind TLR9have been evaluated as cancer vaccine adjuvants and have shown some efficacy in inducing adaptive and antigen-specific, cellular anti-tumor immune responses in animal models. However, early-stage clinical trials in patients with melanoma and gliomas have been less promising. Although higher CpG doses may improveclinical efficacy, significant inflammatory response in an already edematous brain may hinder this approach. Recently, we showed that intratumoral delivery of low-dose, immunostimulatory CpG conjugated with carbon nanotubes (CNTs) both eradicated intracranial (i.c.) gliomas, and induced antitumor immunity that protected mice from subsequent i.c. tumor rechallenge.This paper was divided into three parts:1. Extract glioma stem cells (GSCs) from glioblastoma cell line U251, investigate the expression of HMGA1in GSCs and U251, and then to further demonstrate HMGA1was correlated with malignant of tumors from the prospective of TSCs.2. Explore the expression of HMGA1in malignant gliomas with different WHO classifications and to study the correlation of HMGA1expression with tumor proliferation, invasion and angiogenesis.3. To enhance CpG activity, functionalized carbon nanotubes (CNT) were conjugated with CpG (CNT-CpG) and evaluated in tumor-bearing mice. We tested this hypothesis and demonstrated that intracerebral CNT-CpG may even be superior to systemic therapy, have systemic antitumor immunity to abrogate the growth of both intracerbral and primary tumors.The first part:the different HMGA1expression of total population of glioblastoma cell line U251and glioma stem cells isolated from U251Objective:Extract glioma stem cells (GSCs) from glioblastoma cell line U251, investigate the expression of HMGA1in GSCs and U251, and then to further demonstrate HMGA1was correlated with malignant of tumors from the prospective of TSCs.Methods:Glioma stem cells (GSCs) expressing the surface marker CD133from human glioblastoma cell line U251were isolated using MACS column and were analyzed using immunofluorescence and flow cytometry (FCM). The different expression of HMGA1was detected using real-time quantitative PCR and Western-Blot at transcription and translation levels between U251and isolated GSCs.Results:1, GSCs were successfully isolated from U251and cultured in serum-free medium (SMF). The percentage of GSCs in U251was0.32±0.07%.2, HMGA1expression was significantly higher in GSCs than in glioblastoma cells (P<0.05), up6.13±0.25-fold and2.75±0.99-fold at transcription and translation level, respectively.Conclusion:These results indicated HMGA1is overexpressed in GSCs as compared to glioblastoma cell line U251, which points to the expression of HMGA1being closely related to malignant proliferation, invasion and differentiation of tumor from the prospective of tumor stem cells (TSCs). We conclude that HMGA1may be a potential rational therapeutic target for glioma and GSC. The second part:HMGA1expression in human gliomas and its correlation with tumor proliferation, invasion and angiogenesisObjective:Investigate the expression of HMGA1in malignant gliomas with different WHO classifications and to study the correlation of HMGA1expression with tumor proliferation, invasion and angiogenesis.Methods:Sixty-four tissue specimens including4normal brain tissue samples from brain contusion and laceration patients,22diffuse astrocytomas,23anaplastic astrocytomas and15glioblastomas multiforme were collected at the Provincial hospital affiliated to Shandong University. The expression of HMGA1in malignant gliomas with different WHO classifications and its correlation with tumor proliferation, invasion and angiogenesis was analyzed by using immunohistochemistry and Real-time quantitative PCR.Results:1, Immunohistochemistry results showed that nuclear immunostaining of HMGA1protein was not observed in normal brain tissues, but was observed in96.7%(58of60) of malignant gliomas including high (+++) in15(25.0%), moderate (++) in28(46.7%), negligible to low (0-+) in17(28.3%) samples. The expression of HMGA1protein was significantly higher in glioblastoma multiforme than in WHO grade Ⅱ(P=0.002) and WHO grade Ⅲ gliomas (P=0.024). HMGA1protein expression has significant correlation with expression of Ki-67(r=0.53, P=0.00), MMP-9(r=0.508, P=0.000), VEGF-A (r=0.316, P=0.014) and MVD (r=0.321, P=0.012).2, Real-time quantitative PCR results also supported HMGA1overexpression in glioblastoma multiforme compared to WHO grade Ⅱ(P=0.043) and WHO grade Ⅲ gliomas (P=0.031). HMGA1gene expression has significant correlation with the gene expressions of Ki-67(r=0.429, P=0.025), MMP-9(r=0.443, P=0.024), VEGF-A(r=0.409, P=0.034).Conclusion:These results indicated that the expression of HMGA1has significant correlation with malignancy, proliferation, invasion and angiogenesis of gliomas. We conclude that HMGA1could be used as an intelligent biomarker in diagnosis and treatment of human tumors. The third part:Intracerebral CpG immunotherapy with carbon nanotubes abrogates growth of primary and intracerbral metastasis melanomasObjective:To enhance CpG activity, functionalized carbon nanotubes (CNTs) were conjugated with CpG (CNT-CpG) and evaluated in tumor-bearing mice. We tested this hypothesis and demonstrated that intracerebral CNT-CpG may even be superior to systemic therapy, have systemic antitumor immunity to abrogate the growth of both intracerbral and primary tumors.Methods:In vitro, the transcription factors NF-κB secreted by RAW MP cells was used for measure TLR-9activation. In vivo, the biodistribution of CpG and CNT-CpG were measured with a Xenogen IVIS Imaging System. Cytotoxicity Assay was measured using a Cobra Quantum gamma counter. Flow cytometry and FlowJo8.4.7software were used for measure the proportion of each cell type was measured as percent of total inflammatory cellsResults:1, In vitro experiments, CNT-CpG preparations that increased NF-κB activity at least2-fold, compared to an equivalent dose of free CpG at24hours,2, In vivo experiments, both CNT-CpG-Cy5.5and free CpG-Cy5.5appeared to clear slower from i.c. than s.c. melanomas, and even after7days, some Cy5.5signal was still detectable in the brains. We next compared the local distribution of free CpG to CNT-CpG in i.c. melanomas. Within a few days, free CpG appeared to diffuse away from the injection site, but CNT-CpG dispersed around the tumor margin.3, Compare with free CNT and free CpG, low doses of i.c. CNT-CpG can induce not only a local, but also a systemic anti-tumor response. Although mice survived longer, in contrast to gliomas, they were not cured from i.c. melanomas by i.c. CNT-CpG4, Treatment with i.c. CNT-CpG increased the infiltration of MP (CD45hi CD11b+), NK, CD8, and CD4cells into i.e. tumors, but had no effect on inflammatory cell infiltration into the non-treated s.c. tumors in the same animals. Interestingly, splenocytes from i.c. CNT-CpG- injected mice elicited a stronger ex vivo anti-tumor response when compared to s.c. treated tumors.Conclusion:We have verified the efficacy of a nanoparticle delivery system for optimizing CpG immunotherapy in a mouse melanoma model. More significantly, we have demonstrated potential value of intracerebral CNT-CpG for metastatic brain tumor therapy. Efficient uptake of CNT-CpG by tumor-associated MG in the brain, and their retention and wider distribution around tumors may account for a stronger immune anti-tumor response following intracerebral CNT-CpG injections. CNT-CpG can induce not only a local, but also a systemic anti-tumor response.更多还原