裸鼠原位和异位脑肿瘤生物发光信号成像实验研究

目的应用生物发光成像技术,非侵入性地连续检测活体裸鼠原位和异位脑肿瘤发展演进过程。方法用SMPU-R-MND-luc载体转染人脑肿瘤U87MG细胞系,形成具有高荧光素酶活性的细胞克隆。在裸鼠脑内和胁腰部皮下植入持续表达荧光素酶的肿瘤细胞,建立原位和异位脑肿瘤模型,用影像学资料显示肿瘤部位。用光子发射定量分析动态监测肿瘤生长情况。结果成功地建立了表达荧光素酶活性的原位和异位脑肿瘤动物模型。采集反映肿瘤生长的生物发光信号,肿瘤细胞植入后不同时间点的发光信号值呈显著正相关,而且原位和异位脑肿瘤间存在明显差异。但生物发光脑肿瘤生物发光信号值在第4 d和第14 d时无显著差异。结论体内生物发光成像可以非侵入性地动态检测活体内脑肿瘤演进过程,为研究肿瘤发展机制及最佳治疗策略的选择提供了新的手段和工具。     

恶性胶质瘤的治疗:综合方案的进展

第一部分 中枢神经系统肿瘤（central nerous system tumors） 一、流行病学特征（epidemiological features） 中枢神经系统原发肿瘤，从高度恶性胶质瘤和原始神经外胚层肿瘤到良性脑膜瘤、神经瘤及垂体腺瘤，可表现出不同的生物学行为。根据来自美国脑肿瘤注册中心（Central Brain Tumor Registry of the United States，CBTRUS）的最新报告，每年新诊断的原发脑恶性肿瘤的人数为7．30／10万人。美国脑肿瘤注册中心2005年报告新诊断的中枢神经系统原发肿瘤患者为21690例，死亡12760例。胶质瘤占神经系统原发肿瘤的42％，占恶性肿瘤的77％。胶质瘤起源于不同的组织类型，包括少突胶质细胞瘤、星形细胞瘤、混合型少突星形细胞瘤，表现为不同的恶性程度，而且均具有向高度恶性转化的趋势，其中多形性胶质母细胞瘤（glioblasto mamultiforme，GBM）为成人中最常见的并具有浸润性的原发脑肿瘤。     

副肿瘤综合征的神经系统损害

副肿瘤综合征（Paraneoplasic Syndrome）是在某些恶性肿瘤患者体内，肿瘤未转移的情况下引起的远隔自身器官功能的异常改变。副肿瘤综合征可影响到体内的许多器官和组织，如内分泌、神经肌肉、结缔组织、血液系统和血管，导致相应器官组织异常改变，并非是恶性肿瘤直接转移或浸润所影响的远隔自身器官。其中，发生在神经系统如中枢神经、周围神经、神经肌肉接头或肌肉的病变，称之为神经系统副肿瘤综合征（NPS）。其发生率较低，并因肿瘤类型的不同而异。但由此综合征所造成的损害而出现的临床表现较肿瘤本身更早，也可更为严重，因此，在临床上对此综合征要高度重视，一旦诊断确诊后或高度疑诊此病时，应积极寻找恶性肿瘤的证据。所以，NPS对恶性肿瘤的早期诊断具有重要的临床意义。     

小胶质细胞/巨噬细胞在胶质母细胞瘤中作用的研究进展

胶质母细胞瘤（GBM）是成人最常见的颅内恶性肿瘤,具有极强的侵袭性。此外,由于化疗药物难以通过血脑屏障和胶质母细胞瘤的耐药性,及对放疗敏感性较差等特点,故预后极差。其中肿瘤微环境的改变起到了至关重要的作用,在微环境中胶质瘤相关的小胶质细胞/巨噬细胞（GAMs）的作用正逐渐被重视。GAMs不仅有中枢系统的常驻小胶质细胞,还有来自外周的巨噬细胞。GAMs还有截然不同的两种极化类型,即抑制肿瘤生长的M1表型和促进肿瘤生长的M2表型。并且GAMs不单单和肿瘤细胞具有联系,还与微环境中其他非癌性脑细胞也有互动。该文将从GAMs的来源、极化、与肿瘤微环境中各种细胞间的相互影响阐述其在GBM中的作用,并且从靶向治疗的角度探讨其最新的研究进展。     

大脑淋巴系统的概览及临床意义

传统观念认为大脑是"免疫豁免"器官，对病原体和肿瘤的免疫监视有限。随着研究的逐渐深入，神经学家在中枢神经系统（CNS）中窥探到淋巴系统的踪迹。最近的几项研究证实了脑膜淋巴管的存在，并对其进行了初步描述。脑淋巴管解剖结构和功能的明确，揭开了颅内流体动力学的大致全貌，解答了临床中许多疑问。更重要的是，这些发现为神经系统相关疾病的治疗开辟了新的途径。该文通过回顾中枢淋巴网络的研究历史，总结出大脑组织液（ISF）和脑脊液（CSF）的循环过程，并简要介绍了该淋巴系统在多种疾病的病理生理过程及治疗中的意义。     

U251细胞系来源的脑肿瘤干细胞原位移植瘤动物模型的建立

目的 从U251细胞系中分离脑肿瘤干细胞并以其为移植物建立动物模型.方法 应用悬浮培养法获得脑肿瘤干细胞,用免疫磁珠分选法分离CD133阳性和阴性细胞;将上述3种细胞植入裸鼠颅内.取移植组织切片观察及进行再培养.结果 免疫磁珠分离技术可获得CD133阳性细胞.悬浮培养法所得的细胞和CD133阳性及阴性细胞进行裸鼠原位移植的成瘤率分别为71.4％、80％和0％.结论 U251细胞系中存在脑肿瘤干细胞.裸鼠颅内注射移植脑肿瘤干细胞能够建立肿瘤模型.     

神经干细胞荷载溶瘤病毒靶向调控肿瘤微环境治疗脑胶质母细胞瘤

胶质母细胞瘤(GBM)是临床中最常见的颅内原发性恶性脑肿瘤，预后差、病死率高，目前缺乏有效的治疗手段。作为新型免疫治疗的溶瘤病毒(OV)具有特异性裂解肿瘤细胞的特点，并能够通过调节肿瘤微环境发挥抗肿瘤免疫反应。由于机体预先存在针对病毒的免疫力，OV在肿瘤组织的扩散能力及穿透性不佳等原因限制了其治疗效果。神经干细胞(NSCs)的肿瘤“归巢”特性及在脑实质内良好的迁移能力，可以作为OV的理想载体。临床前研究及Ⅰ期临床试验均表明NSCs荷载OV对GBM具有明显的治疗效果。文中对NSCs荷载OV靶向调控肿瘤微环境，达到治疗GBM的可能和挑战性进行文献学习。     

多形性胶质母细胞瘤的免疫治疗

正多形性胶质母细胞瘤(glioblastoma multiforme,GBM)是一种常见的颅内原发性恶性肿瘤,5年存活率不到10%。目前,GBM的标准治疗包括手术切除加放疗并辅以化疗,但是GBM具有发展迅速、弥漫性浸润、高度恶性的特性,即使接受标准治疗,总生存期仍15个月。免疫治疗是一种独特的治疗方式,以增强抗肿瘤免疫反应和阻断肿瘤免疫逃逸,达到治疗GBM的目的。免疫治疗GBM的临床研究取得令人鼓舞的结果。本文就GBM免疫治疗包括肿瘤疫苗、免疫检查点阻断剂、抗体、T细胞治疗的最     

胶质母细胞瘤分子靶向治疗研究进展

胶质母细胞瘤(glioblastoma,GBM)的分子靶向治疗是目前研究的热点。大量针对GBM进行分子靶向治疗的临床研究相继开展,部分靶向药物如贝伐单抗、西地尼布、西仑吉肽、恩扎妥林和尼妥珠单抗已完成了Ⅲ期临床试验。大部分Ⅲ期临床试验未得出预想的阳性结果 ,然而,研究结果也显示某些特定的亚型能从靶向治疗中获益。未来的研究应充分考虑到GBM的异质性,转变试图以一种靶向药物治疗所有GBM的观念。寻找能预测特定靶向治疗效果的分子标记物,在基因和分子分型的基础上进行个体化的精准靶向治疗。     

骨肉瘤动物模型构建的实验学特点

目前对骨肉瘤的发病及其转移发展以及肿瘤耐药性等仍然了解甚少。通过肿瘤动物模型来对其进行进一步研究，可以更好的推动骨肉瘤治疗。目前常用的骨肉瘤动物模型有小鼠、大鼠、兔、犬等。大鼠器官与人类相似，基因型与人类也相近。犬类可自发骨肉瘤，与人类有一定的相似，常用作自发性肿瘤模型。但肿瘤模型与人类肿瘤在生物学特性、致病机制、组织学等方面尚存在一定差别。随着研究的进一步深入，必将有越来越多符合人类骨肉瘤特点的新型动物模型得到应用。本文主要从骨肉瘤动物模型的建立方法、模型特点及具体应用等方面进行综述。     

The role of CD44 in glioblastoma multiforme

A transmembrane molecule with several isoforms, CD44 is overexpressed in many tumors and promotes tumor formation through interactions with the tumor microenvironment. CD44 has been implicated in malignant processes including cell motility, tumor growth, and angiogenesis. The role of CD44 has been examined in many cancer types. This paper provides, to our knowledge, the first focused review of the role of CD44 in glioblastoma multiforme (GBM), the most common and fatal of primary brain cancers. We summarize research that describes how CD44 promotes GBM aggressiveness by increasing tumor cell invasion, proliferation and resistance to standard chemoradiation therapy. Effects of CD44 inhibition in GBM are also explored. Clinical trials investigating CD44 targeting in CD44-positive solid tumors are underway, and the evidence presented here suggests that CD44 inhibition in GBM may be a promising therapy.     

Applications of organoid technology to brain tumors

Lacking appropriate model impedes basic and preclinical researches of brain tumors. Organoids technology applying on brain tumors enables great recapitulation of the original tumors. Here, we compared brain tumor organoids (BTOs) with common models including cell lines, tumor spheroids, and patient-derived xenografts. Different BTOs can be customized to research objectives and particular brain tumor features. We systematically introduce the establishments and strengths of four different BTOs. BTOs derived from patient somatic cells are suitable for mimicking brain tumors caused by germline mutations and abnormal neurodevelopment, such as the tuberous sclerosis complex. BTOs derived from human pluripotent stem cells with genetic manipulations endow for identifying and understanding the roles of oncogenes and processes of oncogenesis. Brain tumoroids are the most clinically applicable BTOs, which could be generated within clinically relevant timescale and applied for drug screening, immunotherapy testing, biobanking, and investigating brain tumor mechanisms, such as cancer stem cells and therapy resistance. Brain organoids co-cultured with brain tumors (BO-BTs) own the greatest recapitulation of brain tumors. Tumor invasion and interactions between tumor cells and brain components could be greatly explored in this model. BO-BTs also offer a humanized platform for testing the therapeutic efficacy and side effects on neurons in preclinical trials. We also introduce the BTOs establishment fused with other advanced techniques, such as 3D bioprinting. So far, over 11 brain tumor types of BTOs have been established, especially for glioblastoma. We conclude BTOs could be a reliable model to understand brain tumors and develop targeted therapies.     

CX3CL1 and CX3CR1 in the GL261 murine model of glioma: CX3CR1 deficiency does not impact tumor growth or infiltration of microglia and lymphocytes

Human glioblastoma multiforme (GBM) is the most malignant form of human brain tumors. A characteristic of GBM is the marked presence of tumor infiltrated microglia/macrophages and lymphocytes. The goal of this study was directed toward understanding the role of the chemokine system CX3CL1 and its receptor CX3CR1 in the GL261 murine model of malignant glioma. In situ hybridization analysis identified CX3CL1 and CX3CR1 expression in GL261 tumors. The impact of CX3CR1 deletion on the growth of intracranial GL261 gliomas and associated immune cell infiltration was evaluated in CX3CR1 gene-disrupted C57BL/6 mice. A slight increase in the tumor growth rate in CX3CR1-/- mice was evident with similar numbers of microglia and CD4+, CD8+, FoxP3+, or Ly49G2+ lymphocytes within tumors established in CX3CR1 +/- and -/- mice. These data indicate that CX3CR1 has little or no effects on either gliomagenesis or the migration of microglia and lymphocytes into GL261 tumors.     

The involvement of thrombin in the pathogenesis of glioblastoma

Prothrombin and its active derivative thrombin are key members of the coagulation system. The only site of extra‐hepatic thrombin expression is the central nervous system (CNS), where it is involved in brain development, protection, and regeneration. Thrombin affects various degenerative and ischemic CNS diseases like Alzheimer's, multiple sclerosis, cerebral ischemia, and hemorrhage in a dose dependent manner. Additionally, the association of thrombin with various malignancies has recently become evident. Thrombin facilitates the interaction between tumor cells with platelets, endothelial cells, and the adhesion to matrix proteins in various tumor types. Consequently, thrombin enables tumor cell seeding and metastasis, resulting in increased tumor cell growth and angiogenesis. Despite the exceptional position of thrombin in the CNS, its involvement in brain tumor course and development has so far been largely neglected. Over the last decade, several studies found a detrimental effect of thrombin in the most devastating of all primary brain tumors, glioblastomas (GBM). This review highlights the current knowledge on the involvement of thrombin in the pathophysiology and clinical course of GBMs. © 2017 Wiley Periodicals, Inc.     

转染外源性p16基因对恶性胶质瘤生长抑制作用的动物体内实验研究

目的 :探讨p16基因在体内对恶性胶质瘤的生长抑制作用。方法 :将外源性p16基因导入C6 细胞内 ,应用立体定向技术将C6细胞种植于SD大鼠尾状核头部 ,用核磁共振 (MR)扫描技术 ,动态观察颅内肿瘤生长情况。并通过免疫组化、原位杂交和细胞凋亡检测肿瘤细胞的增殖活性。结果 :转染组和治疗组大鼠生存期较对照组明显延长。治疗组肿瘤随时间的延长逐渐缩小。免疫组化显示转染组和治疗组P16蛋白表达明显增强。原位杂交和细胞凋亡检测表明 ,转染组和治疗组大鼠肿瘤细胞增殖活性降低。结论 :p16基因在体内有抑制恶性胶质瘤生长的作用 ;瘤体内注入p16cDNA质粒 脂质体复合物 ,可使肿瘤生长受到明显抑制 ,并使多数肿瘤消失     

9L/Wistar大鼠脑胶质肉瘤模型的建立

目的建立稳定的9L／Wistar大鼠脑胶质肉瘤模型。方法构建稳定表达萤火虫荧光素酶PGL的9L细胞株9L^lvc，采用立体定向手术，在Wistar大鼠右侧尾状核接种DAPI荧光标记的9L^lvc细胞，分别于接种后第7，14，21天通过Xenogen活体动物体内成像系统检测肿瘤的生长情况，并断头取材，观察肿瘤的形态学变化，采用免疫组化的方法检测肿瘤细胞胶质纤维酸性蛋白（GFAP）、波形蛋白（Vemintin）的表达。结果大鼠接种9L^lvc细胞后成瘤率100％。活体成像显示细胞接种后第7天到第21天肿瘤呈逐渐增大趋势。组织切片结果显示肿瘤与周围脑组织边界较清楚，未见包膜；瘤内新生血管丰富，可见出血。肿瘤组织免疫组织化学GFAP及Vemintin蛋白染色阴性。结论该方法建立的大鼠脑胶质肉瘤模型稳定可靠，符合恶性胶质肉瘤的生物学特征．是理想的脑胶质肉瘤实验研究材料。     

Imaging of murine brain tumors using a 1.5 Tesla clinical MRI system

BACKGROUND: In this study, we investigated the feasibility of using a 1.5 Tesla (T) clinical magnetic resonance imaging (MRI) system for in vivo assessment of three histopathologically different brain tumor models in mice. METHODS: We selected mouse models in which tumor growth was observed in different intracranial compartments: Patched+/- heterozygous knock-out mice for tumor growth in the cerebellum (n = 5); U87 MG human astrocytoma cells xenografted to the frontal lobe of athymic mice (n = 15); and F5 (n = 15) or IOMM Lee (n = 15) human malignant meningioma cells xenotransplanted to the athymic mouse skull base or convexity. Mice were imaged using a small receiver surface coil and a clinical 1.5 T MRI system. T1- and fast spin echo T2-weighted image sequences were obtained in all animals. Gadolinium was injected via tail vein to better delineate the intracranial tumors. Twenty mice were followed by serial MRI to study tumor growth over time. In these mice, images were typically performed after tumor implantation, and at two week intervals. Mice were euthanized following their last imaging procedure, and their tumors were examined by histopathology. The histopathological preparations were then compared to the last MR images to correlate the imaging features with the pathology. RESULTS: Magnetic resonance imaging delineated th tumors in the cerebellum, frontal lobes and skull base in all mouse models. The detection of intracranial tumors was enhanced with prio administration of gadolinium, and the limit of resolution of brain tumors in the mice was 1-2 mm3. Sequential images performed at different time intervals showed progressive tumor growth in all animals. The MR images of tumor size and location correlated accurately with th results of the histopathological analysis. CONCLUSION: Magnetic resonance imaging of murine brain tumors in different intracrania compartments is feasible with a 1.5 T clinical MR system and a specially designed surface coil. Tumors as small as 1-2 mm3 can be detecte with good image resolution. Mice harbouring nascent brain tumors can be followed sequentially by serial MR imaging. This may allow for a noninvasive means by which tumor growth can be measured, and novel therapies tested without resorting to sacrifice of the mice.     

Glial TLR2-driven innate immune responses and CD8+ T cell activation against brain tumor

Growing interest has been focused on the roles of microglia as sentinels and effector cells that guard diverse pathological milieu in the brain. Here, it has been reported that microglial TLR2 is a crucial molecule that confers innate and adaptive immunity against brain tumor. TLR2 is preferentially expressed on microglia, brain-resident immune cells, in the tumor-bearing cerebral hemisphere of mouse and rat intracranial tumor models. Microglial TLR2 rapidly responds to brain tumor and modulates the inflammation-associated immune responses including phagocytosis, which are markedly decreased in TLR2-deficient mice. We further reveal that TLR2, but not TLR4, is essential for the tumor-triggered increase of MHC I in microglia. in vitro co-culture and in vivo experiments show that the glial TLR2-MHC I axis contributes to the proliferation and activation of CD8⁺ T cells by brain tumor. In addition, brain tumor-bearing β2m^−/−, Tlr2^−/−, or Rag2 ^−/−γc ^−/− mice exhibit higher tumor volumes compared with WT mice with tumor. Survival analysis of GL26-bearing MHC I-defective mice also support the contribution of glial TLR2-MHC I axis to brain tumor immunity. Moreover, using publicly available data sets of human brain tumor patients, we find that glioblastoma (GBM) tissues with high TLR2 level have similar co-occurrence patterns with MHC I molecules, and the amounts and activity of infiltrating CD8⁺ T cells are correlated with TLR2 level in tissues from GBM patients. Collectively, our findings provide the importance of glial TLR2-driven innate and adaptive immune responses in the brain tumor microenvironment.     

Are gadolinium contrast agents suitable for gadolinium neutron capture therapy?

OBJECTIVE: Gadolinium neutron capture therapy (GdNCT) is a potential treatment for malignant tumors based on two steps: (1) injection of a tumor-specific (157)Gd compound; (2) tumor irradiation with thermal neutrons. The GdNC reaction can induce cell death provided that Gd is proximate to DNA. Here, we studied the nuclear uptake of Gd by glioblastoma (GBM) tumor cells after treatment with two Gd compounds commonly used for magnetic resonance imaging, to evaluate their potential as GdNCT agents. METHODS: Using synchrotron X-ray spectromicroscopy, we analyzed the Gd distribution at the subcellular level in: (1) human cultured GBM cells exposed to Gd-DTPA or Gd-DOTA for 0-72 hours; (2) intracerebrally implanted C6 glioma tumors in rats injected with one or two doses of Gd-DOTA, and (3) tumor samples from GBM patients injected with Gd-DTPA. RESULTS: In cell cultures, Gd-DTPA and Gd-DOTA were found in 84% and 56% of the cell nuclei, respectively. In rat tumors, Gd penetrated the nuclei of 47% and 85% of the tumor cells, after single and double injection of Gd-DOTA, respectively. In contrast, in human GBM tumors 6.1% of the cell nuclei contained Gd-DTPA. DISCUSSION: Efficacy of Gd-DTPA and Gd-DOTA as GdNCT agents is predicted to be low, due to the insufficient number of tumor cell nuclei incorporating Gd. Although multiple administration schedules in vivo might induce Gd penetration into more tumor cell nuclei, a search for new Gd compounds with higher nuclear affinity is warranted before planning GdNCT in animal models or clinical trials.