脑胶质瘤神经干细胞治疗研究进展

脑胶质瘤是神经外科最常见的恶性肿瘤,发病率约占全身肿瘤的5%,占儿童肿瘤的70%,且呈逐年上升的趋势。脑胶质瘤恶性程度高,生长迅速,5年生存率很低,其中高级别胶质瘤具有极强的侵袭能力,目前尚缺乏很有效的根治方法。手术切除肿瘤的难度很大,术后极易复发,预后比较差,对人类健康乃至生命的危害极大。随着分子生物学的发展以及相关生物技术的应用,从基因水平揭示脑胶质瘤的发生发展机制,并寻求有效的基因治疗方法成为人类研究肿瘤治疗新的研究方向。Reynolds和Richards等先后从成年小鼠的纹状体中分离出能够不断增殖且具有多向分化潜能的细胞群,并提出了神经干细胞(neural stem cell,NSC)的概念。NSC具有高度增殖和自我更新的能力,且有迁移功能以及与正常脑组织良好融合的特性,这为基因治疗胶质瘤提供了良好的基础。     

脑胶质瘤动物模型的研究及应用进展

脑胶质瘤约占中枢神经肿瘤的一半,临床治疗效果差。尤其是胶质母细胞瘤,其恶性程度极高,预后性差,是威胁人类健康的主要恶性肿瘤之一,因此,选择一种有效的动物模型是研究脑胶质瘤发病机制及其治疗方法的关键。随着分子生物学、遗传学的发展,尤其是转基因小鼠,以及其他越来越多模式生物的出现,目前已建立了多种脑胶质瘤动物模型。该文将对目前所建立的各种脑胶质瘤动物模型予以综述。     

EGFR和Ki-67在胶质瘤中表达的研究进展

胶质瘤是颅内常见的原发恶性肿瘤,而某些癌基因的激活、过表达或扩增、重排导致脑胶质瘤的形成。主要讨论脑胶质瘤的生物学特点,指出当前研究某些与肿瘤增殖活性及侵袭能力相关的基因改变、蛋白表达,推测其增殖和侵袭活动的具体过程,这是攻克脑胶质瘤的基础和关键。在众多与肿瘤相关的蛋白和基因中,选择了主要反映脑胶质瘤增殖活性和侵袭能力的相关基因——EGFR、Ki-67进行综述。从分子生物学水平评估脑胶质瘤细胞增殖和侵袭状态,在分析和判断胶质瘤的生长、分化程度,指导治疗方案的选择及预后的判断等方面有着重要的实用价值;但对于患者的预后,还需结合年龄、肿瘤位置、病理分级以及其他标记物等进行综合评价。     

miR－155与胶质瘤的研究进展

人脑胶质瘤是最常见的原发性脑肿瘤,起源于脑部神经胶质细胞,约占所有颅内肿瘤的45％左右,在儿童恶性肿瘤中排第二位。胶质瘤系浸润性生长物,它和正常脑组织没有明显界限,难以完全切除,对放疗化疗不甚敏感,非常容易复发,且手术难以切除或根本不能手术。化学药物和一般抗肿瘤的中药,因血脑屏障等因素的影响,疗效也不理想,因此脑胶质瘤至今仍是全身肿瘤中预后最差的肿瘤之一。人类miR-155是由位于21号染色体的BIC基因外显子3编码的多功能miRNA,在干细胞分化、免疫、炎症、癌症、心血管疾病以及病毒感染的病理生理过程中发挥重要作用,也是联系炎症和癌症的桥梁。miR-155在人胶质瘤中作用机制的研究才刚刚起步,目前miR－155与人胶质瘤的关系的研究已成为研究热点。人胶质瘤中miR-155及其相关调控机制的研究。将更有利于人胶质瘤的早期诊断和基因治疗的发展。本文就miR-155与胶质瘤的研究进展予以综述。     

shRNA干扰eIF4A1基因对胶质瘤细胞体内外生物学特征的影响

胶质瘤是颅内常见的肿瘤,其中恶性脑胶质瘤成弥漫性生长,尽管给予手术、放疗或化疗等综合治疗,仍极易复发,迫切需要探索新的治疗方法.但是胶质瘤的治疗靶点匮乏,因此探索有效的靶点对其治疗具有重要的意义.本研究中首先利用中国脑胶质瘤基因组图谱计划数据库(Chinese glioma genome altas, CGGA)分析了真核生物翻译起始因子中eIF4A1与脑胶质瘤的关系.生物信息学分析显示, eIF4A与胶质瘤病理分级相关并且在胶质瘤细胞中高表达. eIF4A1的抑制剂Silvestrol明显抑制胶质瘤细胞的增殖能力.利用慢病毒感染胶质瘤细胞建立shRNA干扰eIF4A1基因的胶质瘤细胞株,进行了一系列体内外生物学特征的实验,研究结果发现, shRNA干扰eIF4A1基因后能够有效抑制胶质瘤细胞的增殖、克隆形成、细胞侵袭和迁移.因此,本实验研究证实eIF4A1是胶质瘤治疗的有效靶点,为胶质瘤的临床治疗提供可靠的理论基础.     

MMP-9和PTEN在胶质瘤中的表达及相关性分析

目的：检测脑胶质瘤组织中MMP-9和PTEN表达情况并探讨其意义。方法：采用免疫组织化学S-P法检测50例脑胶质瘤、10例正常脑组织中两者的表达。结果：脑胶质瘤组织中MMP一9和PTEN表达定位于细胞浆,其阳性表达率分别为74．00％（37／50）和60．00％（30／60）与正常脑组织0、100％有差异（P〈0．05）。两者的表达与胶质瘤患者的年龄、性别无明显相关性（P〉O．05）,但高级别组与低级别组之间表达有差异,具有统计学意义（P〈0．05）;在脑胶质瘤组织中MMP-9蛋白的阳性表达与PTEN蛋白的阳性表达有相关性,二者呈负相关（P〈0．05）。结论：MMP．9蛋白在胶质瘤组织中的表达明显高于正常脑组织,并且高级别胶质瘤组的表达明显高于低级别胶质瘤组中的表达,提示MMP-9可能与胶质瘤的浸润、发展、转移有关,可作为一个预测胶质瘤侵袭转移能力的肿瘤标志物。PTEN蛋白在正常脑组织中的表达明显高于胶质瘤,且高级别组表达明显高于低级别组,提示PTEN基因突变或缺失在胶质瘤的发生发展中起重要作用,且与肿瘤恶性分化程度密切相关,表明PTEN的失活预示着一个特殊的进展性的临床行为,在胶质瘤恶性进展中属于较晚发生的分子事件。     

人跨模型和跨膜稳定型肿瘤坏死因子-α的基因表达产物对体外培养的脑胶质瘤细胞的影响

为了探讨人跨膜型和跨膜稳定型肿瘤坏死因子 - α(TNF- α)对脑恶性胶质瘤基因治疗的有效方法 ,研究应用了基因重组、细胞培养以及免疫组化等技术 ,通过真核表达载体 pc DNA3转染 NIH3T3细胞 ,并在体外观察了 TNF- α对脑胶质瘤生长的影响。结果显示 :与对照组相比 ,TM- TNF- α组和 TM- TNF- αm组中 GFAP和 S- 10 0免疫反应阳性肿瘤细胞的数量、胞体截面积、周长、平均光密度皆明显减少。统计学分析提示差异有显著性意义。这表明 TNF- α基因的表达可明显抑制胶质细胞的生长和分化。而且 ,还发现跨膜稳定型 TNF- α的杀瘤效果较跨膜型 TNF- α强。这为 TNF- α基因用于胶质瘤的基因治疗提供了一定的实验依据 ,提示大剂量的 TNF- α在临床治疗可能产生毒副作用。     

慢病毒介导EGFP转染骨髓间充质干细胞在胶质瘤模型中迁移分布的研究

目的:如何证实骨髓间充质干细胞(BMSCs)是胶质瘤基因治疗中最好的药物载体?将增强型绿色荧光蛋白(EGFP)标记的大鼠骨髓间充质干细胞移植入大鼠C6胶质瘤模型脑内,观察骨髓间充质干细胞在肿瘤内的迁徙与定位。方法:贴壁法培养大鼠骨髓细胞获取纯化的BMSCs。慢病毒介导EGFP转染BMSCs,于荧光显微镜下观察EGFP的表达,并行流式细胞仪检测EGFP阳性转染率。利用立体定向仪将培养好的C6细胞注入大鼠脑内,建立大鼠脑内胶质瘤模型。将标记EGFP的BMSCs利用微量注射器注入模型鼠脑内;移植后第1,7天处死大鼠,用荧光显微镜观察BMSCs在肿瘤内的迁移分布。结果:实验成功建立了大鼠脑内胶质瘤模型。以EGFP标记的BMSCs在模型鼠脑内主动迁移分布于肿瘤内部及肿瘤与正常脑组织交界侧。结论:骨髓间充质干细胞可以作为肿瘤基因治疗的良好载体。     

SD大鼠与Wistar大鼠脑胶质瘤动物模型的建立及比较

目的比较利用SD大鼠、Wistar大鼠建立脑胶质瘤动物模型的不同,为研究脑胶质瘤的发病机制及治疗方法提供操作平台。方法利用立体定向仪建立SD大鼠、Wistar大鼠大脑皮层接种C6细胞（2.5×105个细胞/只）,建立脑胶质瘤动物模型,利用组织病理学、免疫组织化学以及核磁共振成像等技术,比较两种动物模型在成瘤率、肿瘤生长状况、死亡率以及动物一般情况等方面的异同。结果SD大鼠组、Wistar大鼠组的成瘤率均为100%,两组均未见转移;但SD大鼠组肿瘤成瘤时间较长,且部分肿瘤有自愈倾向,而Wistar大鼠组则未出现类似情况。结论Wistar大鼠大脑皮层脑胶质瘤动物模型的肿瘤性状更接近于人的脑胶质瘤,因此更适合探索和研究脑胶质瘤的发病机制和治疗方法;而SD大鼠的肿瘤由于性状类似转移瘤,且有自愈倾向,不适合作为上述相关研究的动物模型。     

苹果酸酶3(ME3)对人脑胶质瘤细胞的增殖、迁移、侵袭及间质转换的影响

该研究探讨了苹果酸酶3(malic enzyme 3,ME3)对人脑胶质瘤细胞增殖、迁移、侵袭和间质转换能力的影响。首先,用Real-time PCR和Western blot检测胶质瘤细胞中ME3的m RNA及其蛋白质的水平。使用质粒(sh-ME3)转染高表达ME3的脑胶质瘤细胞U87MG和U251MG,CCK-8和克隆形成实验分别检测细胞增殖以及克隆形成能力。Transwell实验检测细胞迁移和侵袭能力,并且采用细胞划痕实验进一步检测细胞的迁移能力。用Western blot检测干扰ME3后细胞间质表型标志物。结果表明,ME3下调后,U87MG和U251MG细胞的增殖、迁移和侵袭能力减弱,并且胶质瘤间质表型标志物的表达明显降低,间质转换能力被抑制。以上结果说明,ME3在脑胶质瘤细胞的增殖、迁移和侵袭中发挥了重要作用,同时,ME3因其在胶质瘤发展中发挥的重要作用可能成为肿瘤治疗的新靶点。     

Migratory neural stem cells for improved thymidine kinase-based gene therapy of malignant gliomas

Gene therapy of glioma based on viral delivery of herpes simplex virus type I thymidine kinase (HSV-TK) has failed in the clinic because of low transduction efficacy. To circumvent this problem, this study evaluated highly migratory HSV-TK-transduced neural stem cells (NSC) for their ability to kill untransduced glioma cells by a gap junction-mediated bystander effect. The admixture of HSV-TK-transduced NSC to U87MG and LN-18 human malignant glioma cell lines at ratios of 1:10 or 1:1 eliminated more than 50% or 90% of glioma cells in the presence of ganciclovir (25 microM). Glioma cell cytotoxicity required cell-cell contact. Similarly, tumor cell cytotoxicity was observed in two of three primary glioblastoma cell cultures, and the presence of this bystander effect correlated with the expression of connexin 43 in the untransduced glioma target cells. In conclusion, we delineate a role for migratory HSV-transfected NSC to eliminate glioma cells purely by means of the bystander effect.     

Brain Tumor Stem Cells

Primary malignant brain cancer, one of the most deadly diseases, has a high rate of recurrence after treatment. Studies in the past several years have led to the hypothesis that the root of the recurrence may be brain tumor stem cells (BTSCs), stem-like subpopulation of cells that are responsible for propagating the tumor. Current treatments combining surgery and chemoradiotherapy could not eliminate BTSCs because these cells are highly infiltrative and possess several properties that can reduce the damages caused by radiation or anti-cancer drugs. BTSCs are similar to NSCs in molecular marker expression and multi-lineage differentiation potential. Genetic analyses of Drosophila CNS neoplasia, mouse glioma models, and human glioma tissues have revealed a link between increased NSC self-renewal and brain tumorigenesis. Furthermore, data from various rodent models of malignant brain tumors have provided compelling evidence that multipotent NSCs and lineage-restricted neural progenitor cells (NPCs) could be the cell origin of brain tumors. Thus, the first event of brain tumorigenesis might be the occurrence of oncogenic mutations in the stem cell self-renewal pathway in an NSC or NPC. These mutations convert the NSC or NPC to a BTSC, which then initiates and sustains the growth of the tumor. The self-renewal of BTSCs is controlled by several evolutionarily conserved signaling pathways and requires an intact vascular niche. Targeting these pathways and the vascular niche could be a principle in novel brain tumor therapies aimed to eliminate BTSCs.     

Differentiation profile of brain tumor stem cells： a comparative study with neural stem cells

Understanding of the differentiation profile of brain tumor stem cells （BTSCs）, the key ones among tumor cell population, through comparison with neural stem cells （NSCs） would lend insight into the origin of glioma and ultimately yield new approaches to fight this intractable disease. Here, we cultured and purified BTSCs from surgical glioma specimens and NSCs from human fetal brain tissue, and further analyzed their cellular biological behaviors, especially their differentiation property. As expected, NSCs differentiated into mature neural phenotypes. In the same differentiation condition, however, BTSCs exhibited distinguished differences. Morphologically, cells grew flattened and attached for the first week, but gradually aggregated and reformed floating tumor sphere thereafter. During the corresponding period, the expression rate of undifferentiated cell marker CD 133 and nestin in BTSCs kept decreasing, but 1 week later, they regained ascending tendency. Interestingly, the differentiated cell markers GFAP and β-tubulinlII showed an expression change inverse to that of undifferentiated cell markers. Taken together, BTSCs were revealed to possess a capacity to resist differentiation, which actually represents the malignant behaviors of glioma.     

Brain tumour stem cells: implications for cancer therapy and regenerative medicine

The cancer relapse and mortality rate suggest that current therapies do not eradicate all malignant cells. Currently, it is accepted that tumorigenesis and organogenesis are similar in many respects, as for example, homeostasis is governed by a distinct sub-population of stem cells in both situations. There is increasing evidence that many types of cancer contain their own stem cells: cancer stem cells (CSC), which are characterized by their self-renewing capacity and differentiation ability. The investigation of solid tumour stem cells has gained momentum particularly in the area of brain tumours. Gliomas are the most common type of primary brain tumours. Nearly two-thirds of gliomas are highly malignant lesions with fast progression and unfortunate prognosis. Despite recent advances, two-year survival for glioblastoma (GBM) with optimal therapy is less than 30%. Even among patients with low-grade gliomas that confer a relatively good prognosis, treatment is almost never curative. Recent studies have demonstrated the existence of a small fraction of glioma cells endowed with features of primitive neural progenitor cells and a tumour-initiating function. In general, this fraction is characterized for forming neurospheres, being endowed with drug resistance properties and often, we can isolate some of them using sorting methods with specific antibodies. The molecular characterization of these stem populations will be critical to developing an effective therapy for these tumours with very dismal prognosis. To achieve this aim, the development of a mouse model which recapitulates the nature of these tumours is essential. This review will focus on glioma stem cell knowledge and discuss future implications in brain cancer therapy and regenerative medicine.     

Therapeutic effect of genetically modified human neural stem cells encoding cytosine deaminase on experimental glioma

The aim of this study was to determine the efficacy of neural stem cell-based suicidal gene therapy in rats bearing human glioma. F3 human neural stem cells (NSCs) were transduced to encode cytosine deaminase (CD) which converts 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU). Intratumoral or intravenous transplantation of F3.CD human NSCs led to marked reduction in tumor burden and significantly prolonged the survival of brain tumor-bearing rats. The systemic administration of 5-FC with direct intratumoral/intravenous transplantation of F3.CD cells had remarkable therapeutic effect in rats with human glioma cells as compared with transplantation of parental F3 cells. There was 74% reduction in tumor volume in rats receiving direct transplantation of F3.CD cells into tumor site, and 67% reduction in tumor volume in rats receiving intravenous injection of F3.CD cells as compared to control animals transplanted with human glioma U373 cells alone. The combination of F3.CD and 5-FC was a highly effective in the glioma rat model. Our observations suggest that genetically engineered NSCs encoding suicide gene CD could provide clinical application of suicide gene therapy for patients with glioma.     

Glioma stem cells: a midterm exam

Several years ago, the discovery of a highly tumorigenic subpopulation of stem-like cells embedded within fresh surgical isolates of malignant gliomas lent support to a new paradigm in cancer biology--the cancer stem cell hypothesis. At the same time, these "glioma stem cells" seemed to resolve a long-standing conundrum on the cell of origin for primary cancers of the brain. However, central tenets of the cancer stem cell hypothesis have recently been challenged, and the cellular origins of stem-like cells within malignant glioma are still contended. Here, we summarize the issues that are still in play with respect to the cancer stem cell hypothesis, and we revisit the developmental origins of malignant glioma. Do glioma stem cells arise from developmentally stalled neural progenitors or from dedifferentiated astrocytes? Five separate predictions of a neural progenitor cell of origin are put to the test.     

Getting the right stuff： controlling neural stem cell state and fate in vivo and in vitro with biomaterials

Stem cell therapy holds great promises in medical treatment by, e.g., replacing lost cells, re-constitute healthy cell populations and also in the use of stem cells as vehicles for factor and gene delivery. Embryonic stem cells have rightfully attracted a large interest due to their proven capacity of differentiating into any cell type in the embryo in vivo. Tissue-specific stem ceils are however already in use in medical practice, and recently the first systematic medical trials involving human neural stem cell （NSC） therapy have been launched. There are yet many obstacles to overcome and procedures to improve. To ensure progress in the medical use of stem cells increased basic knowledge of the molecular mechanisms that govern stem cell characteristics is necessary. Here we provide a review of the literature on NSCs in various aspects of cell therapy, with the main focus on the potential of using biomaterials to control NSC characteristics, differentiation, and delivery. We summarize results from studies on the characteristics of endogenous and transplanted NSCs in rodent models of neurological and cancer diseases, and highlight recent advancements in polymer compatibility and applicability in regulating NSC state and fate. We suggest that the development of specially designed polymers, such as hydrogels, is a crucial issue to improve the outcome of stem cell therapy in the central nervous system.     

Stem Cell Markers in Gliomas

Gliomas are the most common tumours of the central nervous system (CNS) and a frequent cause of mental impairment and death. Treatment of malignant gliomas is often palliative because of their infiltrating nature and high recurrence. Genetic events that lead to brain tumours are mostly unknown. A growing body of evidence suggests that gliomas may rise from cancer stem cells (CSC) sharing with neural stem cells (NSC) the capacity of cell renewal and multipotency. Accordingly, a population of cells called “side population” (SP), which has been isolated from gliomas on the basis of their ability to extrude fluorescent dyes, behaves as stem cells and is resistant to chemotherapeutic treatments. This review will focus on the expression of the stem cell markers nestin and CD133 in glioma cancer stem cells. In addition, the possible role of Platelet Derived Growth Factor receptor type α (PDGFR-α) and Notch signalling in normal development and tumourigenesis of gliomas are also discussed. Future work elucidating the mechanisms that control normal development will help to identify new cancer stem cell-related genes. The identification of important markers and the elucidation of signalling pathways involved in survival, proliferation and differentiation of CSCs appear to be fundamental for developing an effective therapy of brain tumours. Special issue article in honor of Dr. Anna Maria Giuffrida-Stella.