CD133鉴定胶质瘤干细胞血管周围壁龛的实验研究

目的 检测脑肿瘤干细胞(BTSC)标记物CD133、巢蛋白(Nestin)和增殖细胞核抗原(PCNA)在74例脑胶质瘤标本中的表达,探讨肿瘤干细胞生存的微环境一壁龛的组成、形态及其在脑肿瘤组织中的分布. 方法 选取安徽医科大学附属省立医院神经外科自2007年1月至2008年10月间手术切除的74例胶质瘤标本,按照WH02000年的神经系统肿瘤分类分级标准分为Ⅱ级22例(低级别组)、Ⅲ级27例和Ⅳ级25例(高级别组),采用免疫组织化学染色和免疫荧光双标法分别检测标本中CD133的表达及其与Nestin、PCNA的共表达情况.计算并比较不同级别胶质瘤组织CD133+细胞、CD133+血管和CD133+壁龛所占的百分比.并对CD133+血管和CD133+壁龛的表达进行相关性分析. 结果 CD133+细胞聚集于壁龛内生长,低级别组胶质瘤中CD133+壁龛阳性率较低.壁龛内增殖细胞较少,与相邻肇龛之间界限清晰,周围CD133+血管分布较少.高级别组胶质瘤中CD133+壁龛阳性率高,壁龛之间无明显界限,壁龛内细胞增殖活跃,周围可见丰富的CD133+血管分布:壁龛中除CD133+/Nestin+BTSC外,可见CD133+/Nestin-细胞、CD133/PCNA+细胞等亚群细胞;不同级别胶质瘤CD133+细胞、CD133+血管、CD133+壁龛百分比不同,且肿瘤级别越高,三者表达越高,差异有统计学意义(P<0.05).CD133+壁龛与CD133+血管的表达呈正相关(r=0.425,P=0.000). 结论 在脑胶质瘤组织中存在着由CD133+/Nestin+BTSC和一些亚群细胞组成的壁龛结构,CD133+血管对于壁龛结构的维持起着非常重要的作用.     

人星形细胞瘤TGF-α表达与血管形成的研究

目的探讨TGF-α在人脑星形细胞瘤中表达,研究其在肿瘤血管形成中的作用。方法用SP免疫组化法检测50例人脑星形细胞瘤标本中TGF-α蛋白表达;用抗CD34相关抗原单克隆抗体免疫组化染色显示肿瘤组织微血管,并以微血管密度(microvesseldensity,MVD)测定肿瘤血管生成。结果实验组50例人脑星形细胞瘤TGF-α表达总阳性率64%(3250);总MVD38.88±19.13,显著高于正常脑组织组9.30±3.74(P<0.01)。随着人脑星形细胞瘤级别增高,MVD数值呈现增高趋势。TGF-α与MVD呈显著线性正相关性(r=0.869,P<0.01))。结论TGF-α在人脑星形细胞瘤中高表达,可能参与人脑星形细胞瘤的血管形成。     

人星形细胞瘤TGF—α表达与血管形成的研究

目的探讨TCF-α在人脑星形细胞瘤中表达，研究其在肿瘤血管形成中的作用。方法用SP免疫组化法检测50例人脑星形细胞瘤标本中TCF-α蛋白表达；用抗CD34相关抗原单克隆抗体免疫组化染色显示肿瘤组织微血管，并以微血管密度（miemvessel density，MVD）测定肿瘤血管生成。结果实验组50例人脑星形细胞瘤TGF-α表达总阳性率64％（32／50）；总MVD38.88±19．13，显著高于正常脑组织组9．30±3．74（P〈0．01）。随着人脑星形细胞瘤级别增高，MVD数值呈现增高趋势。TGF-α与MVD呈显著线性正相关性（r=0．869，P〈0．01））。结论TGF-α在人脑星形细胞瘤中高表达，可能参与人脑星形细胞瘤的血管形成．     

脑肿瘤干细胞在不同病理级别胶质瘤中分布特性

目的 研究不同病理级别的胶质瘤肿瘤干细胞CD133的表达及其分布规律.方法 对78例Ⅰ-Ⅳ脑胶质细胞瘤的病理切片进行CD133免疫荧光染色和免疫组化染色,观察和比较小同病理级别肭胶质细胞瘤肿瘤干细胞之间的分布特点.结果 脑肿瘤干细胞在不同病理级别的胶质瘤石蜡组织切片上CD133阳性表达,在低级别脑胶质细胞瘤中表达很低,以零星分布的存在,未见脑肿瘤干细胞集聚现象;在Ⅲ、Ⅳ胶质细胞瘤组织切片上可以见到数个细胞聚集在一起,在髓母细胞瘤尤为明显,而且Ⅱ～Ⅳ级胶质细胞瘤可以见到多数是围绕着血管或在血管旁,呈巢状分布的均近血管边缘.结论 随着胶质瘤病理级别的升高,肿瘤干细胞分布从低级别的零星分布至高级别的聚集、巢状分布于血管周围.     

脑肿瘤干细胞的增殖活性与微血管密度的相关性研究

目的 探讨脑肿瘤干细胞(BTSC)增殖活性与微血管密度的相关性.方法 采用免疫组织化学染色检测BTSC标记物CD133、增殖细胞核标记物Ki-67和血管内皮细胞标记物CD31在胶质瘤组织中的表达;采用免疫荧光双染法检测CD133/Ki-67和CD133/CD31的共表达情况;并对BTSC的增殖活性与微血管密度的相关性行统计学分析.结果 免疫组化显示:低级别胶质瘤中,CD133、CD31和Li-67的阳性表达较少;而在高级别胶质瘤中,CD133、CD31和Ki-67阳性表达明显增加.免疫荧光双染显示:随着胶质瘤级别升高,CD133/Ki-67共表达明显增加,且可见CD133/CD31共表达于血管内皮细胞.在胶质瘤组织中,Ki-67、CD31表达均与CD133表达呈正相关(n=0.758,P<,1>=0.000;r<,2>=0.439,P<,2>=0.000),ki-67与CD31的表达也呈正相关(r<,3>=0.816,P<,3>=0.000).结论 BTSC与微血管联系密切,不仅在区域分布上围绕微血管增殖分化.并且在功能上与微血管相互促进和依赖.     

人脑胶质瘤干细胞分化为血管内皮细胞的实验研究

目的 探讨体内外原代培养的人脑胶质瘤干细胞(GSCs)与胶质瘤新生血管内皮细胞的关系.方法 新鲜高级别(WHOⅢ级、Ⅳ级)的人脑胶质瘤标本经原代培养获取GSCs,免疫组化法检测其肿瘤干细胞及干细胞标记物Nestin的表达;鉴定后的GSCs经低氧诱导,免疫荧光法检测其诱导分化后内皮细胞标记物CD31、CD144和胶质瘤细胞标记物GFAP的表达,RT-PCR和Western-blot法检测其CD31的表达;建立胶质瘤干细胞皮下荷瘤裸鼠模型,免疫组化技术检测模型中人来源的CD31的表达.结果 (1)悬浮生长的胶质瘤干细胞球样细胞经免疫组化鉴定Nestin表达阳性;(2)低氧诱导后的GSCs能够表达CD31、CD144,有些细胞能够同时表达CD144和GFAP;(3)RT-PCR检测发现GSCs在诱导前后都有CD31 mRNA的表达,而Western-blot检测到只有诱导后的GSCs有CD31蛋白的表达;(4)胶质瘤干细胞荷瘤裸鼠模型的肿瘤组织中部分微血管抗人CD31抗体染色阳性.结论 胶质瘤干细胞不仅在体外低氧条件下可分化为内皮细胞,在体内微环境条件下同样可分化为血管内皮细胞,并参与胶质瘤新生组织的血液供应.     

Stathmin在脑胶质瘤血管内皮细胞中的表达及意义

目的 观察Stathmin及CD105在脑胶质瘤血管内皮细胞中的表达,探讨其在脑胶质瘤血管形成中的作用. 方法 用SP免疫组化法检测10例正常脑组织和78例脑胶质瘤血管内皮细胞中Stathmin和CD105蛋白表达,并通过检测肿瘤微血管密度(MVD)分析肿瘤血管形成.结果 正常脑组织血管内皮细胞中Stathmin和CD105均无表达,脑胶质瘤血管内皮细胞中二者均呈高表达;随着胶质瘤病理级别的增高,Stathmin和CD105表达上调,MVD值增高,Stathmin-MVD和CD105-MVD与脑胶质瘤病理分级均成正相关(r=0.912,P<0.05;P<0.936,P<0.05);且Stathmin-MVD和CD105-MVD之间也存在正相关(r=0.996,P<0.05). 结论 Stathmin与CD105在脑胶质瘤血管内皮细胞中均呈高表达,在肿瘤微血管形成过程起重要作用.     

利拉鲁肽促进实验性脑梗死小鼠血管新生及其作用机制的研究

目的探讨利拉鲁肽(LIRA)对小鼠局灶性脑缺血后血管新生和星形胶质细胞活化的影响。方法采用电凝法制备小鼠局灶性皮质梗死模型(dMCAO)。采用免疫荧光方法测定微血管密度、内皮细胞增殖以及内皮细胞星形胶质细胞包裹率,利用RT-qPCR方法测定VEGF mRNA的表达。结果在dMCAO后,与生理盐水组相比,大剂量LIRA组神经功能评分明显改善,梗死体积明显减小,微血管密度、内皮细胞增殖和内皮细胞星形胶质细胞包裹率显著增加,同时增加VEGF mRNA的表达(P0.05)。结论 LIRA可能通过增加VEGF mRNA的表达促进血管新生和星形胶质细胞活化,从而促进脑梗死后神经功能恢复。     

VEGF在人脑胶质瘤中的表达与肿瘤增殖及肿瘤血管生成的关系

目的研究血管内皮细胞生长因子(vascular endothelial growth factor,VEGF)在人脑胶质瘤中的表达与肿瘤增殖及血管生成的关系。方法应用免疫组化技术和形态定量分析法,检测98例手术切除脑胶质瘤中VEGF表达、PCNA标记指数(PCNA LI)、微血管密度(Microvessel density,MVD)表达。结果(1)肿瘤细胞及血管内皮细胞均可以表达VEGF,阳性颗粒分布于肿瘤胞浆中;(2)高级别肿瘤PCNA、LI、MVD显著高于低级别肿瘤(P<0.05);VEGF表达阳性肿瘤的PCNA、LI、MVD显著高于VEGF表达阴性肿瘤(P<0.05);(3)在星形细胞肿瘤中,随着MVD的增大,VEGF在肿瘤血管内皮的染色率逐渐增加,与肿瘤的MVD存在正相关关系(r值为0.44,P<0.01)。结论脑胶质瘤的VEGF表达与MVD呈正相关关系,VEGF在肿瘤细胞增殖及血管再生过程中起重要作用。     

基于模糊C均值聚类的人脑星形细胞肿瘤病理分级方法初探

目的采用反映血管新生状态的指标经模糊C均值聚类对星形细胞肿瘤病理学分级进行探讨。方法采用含有正常成人脑组织、弥漫性星形细胞瘤（WHOⅡ级）、间变性星形细胞瘤（WHOⅢ级）、胶质母细胞瘤（WHOⅣ级）及阳性对照组织的168点矩阵的组织芯片,通过免疫组织化学SABC双标法标记内皮细胞和血管内皮生长因子,以Image-Pro Plus 5.1中文版图像分析软件对染色结果及血管内皮生长因子阳性单位、微血管密度及微血管平均周长等指标进行测定。采用单因素分析方法筛选与星形细胞肿瘤病理级别相关的参数,以矩阵实验室数学软件提供的模糊C均值聚类函数参数作为聚类对象,将不同的组织切片参数值进行模糊C均值聚类,所得聚类值分别赋值为星形细胞肿瘤病理分级值。结果（1）在不同病理分级组之间,星形细胞肿瘤血管内皮生长因子阳性单位差异具有统计学意义（P=0.000）,各病理分级组间两两比较差异亦有统计学意义（均P〈0.05）。（2）在不同病理分级组之间,星形细胞肿瘤微血管密度值差异有统计学意义（P=0.000）,两两比较差异亦有统计学意义（均P=0.000）。（3）星形细胞肿瘤微血管平均周长,Ⅱ级组与Ⅲ级组、Ⅱ级组与Ⅳ级组比较差异有统计学意义（均P=0.000）,而Ⅲ级组与Ⅳ级组之间差异无统计学意义（P=1.000）。（4）与WHO病理分级相比,模糊C均值聚类产生的星形细胞肿瘤病理分级值对Ⅱ、Ⅲ、Ⅳ级等级别的诊断符合率分别为85.71%、48.39%和78.95%,总体正确率达68.46%。结论星形细胞肿瘤血管内皮生长因子阳性单位、微血管密度和微血管平均周长等项指标的模糊C均值聚类值与星形细胞肿瘤病理分级值比较符合,可应用模糊C均值聚类法对星形细胞肿瘤的病理分级进行辅助推测。     

VR1-positive primary afferents contact NK1-positive spinoparabrachial neurons

Neurons in rat superficial dorsal horn that express neurokinin receptor 1 (NK1), a receptor for substance P, play a critical role in the development of hyperalgesia. Thermal hyperalgesia is dramatically reduced after ablation of these neurons, but, paradoxically, not in mice that lack the NK1 receptor (Mantyh et al. [1997] Science 278:275-279). Because primary afferents that express vanilloid receptor 1 (VR1), a receptor for noxious heat, are essential for thermal nociception and hyperalgesia, we reasoned that VR1-positive fibers may terminate onto NK1-expressing dorsal horn neurons. We therefore combined immunofluorescent staining for VR1 and NK1 to show that NK1-positive neurons in lamina I are contacted by VR1-positive fibers. That these contacts represent synapses was verified by staining for the presynaptic marker synaptophysin and by electron microscopy. By combining retrograde tracing with immunocytochemistry, we also found that most NK1-positive cells contacted by VR1-positive fibers project to the lateral parabrachial nucleus. Because quantitative evaluation suggests a preferential targeting of NK1-positive lamina I neurons by fibers containing VR1, these results demonstrate a significant monosynaptic innervation of spinoparabrachial neurons by VR1-positive afferents.     

NG2-positive cells generate A2B5-positive oligodendrocyte precursor cells

Cellular specification of the oligodendrocyte lineage occurs through a series of stages identified by expression of distinct biochemical characteristics. The best characterized oligodendrocyte progenitor cell (OPC) in vitro is the bipotential O2-A progenitor, identified by labeling with monoclonal antibody A2B5, which proliferates predominantly in response to platelet derived growth factor (PDGF). The cellular ancestors of O2-A progenitor cells are currently unclear. In vivo OPCs can be identified by expression of the cell surface markers NG2 (a sulfated proteoglycan) and platelet derived growth factor receptor alphaR). Substantial evidence supports the generation of oligodendrocytes from NG2(+), PDGFalphaR(+) cells both in vivo and in vitro. The developmental relationship between NG2(+) cells and A2B5(-) positive cells is unknown and it is unclear whether they represent identical, partially overlapping or nonoverlapping populations of cells. Here we show that in cultures of developing brain NG2(+) and A2B5(+) cells arise from overlapping cell populations. NG2(+) cells appear prior to the expression of A2B5(+) cells and generate A2B5(+) cells. We propose that during development NG2(+)/A2B5(-) cells (pre-OPCs) represent the direct ancestor to A2B5(+) O2A progenitor cells (OPCs).     

Lymphocyte subsets in HLA-DR2-positive narcoleptic patients

On the basis of our recent finding that all narcoleptic patients were HLA-DR2 positive, peripheral blood lymphocyte subsets were examined in 30 HLA-DR2 positive narcoleptic patients by using monoclonal antibodies and a flow cytometry. The percentages of OKIa1+ cells and OKM1+ cells increased significantly, while no quantitative changes were observed in the T cell subsets examined in the present study. No major immunological abnormalities which altered the T cell subpopulations quantitatively were apparent in narcolepsy.     

The long-term outcome of anti-Jo-1-positive inflammatory myopathies

Abstract. Objective: To determine the response to treatment and the long-term outcome of patients with the antisynthetase syndrome associated with anti-Jo-1-antibodies. Patients and Methods: A total of 12 patients with histologically proven myositis and anti-Jo-1-autoantibodies were evaluated over a mean follow-up period of 66.4 months. In all patients neuromuscular function tests, electromyographic examinations, pulmonary function tests and high-resolution-computed tomography of the lungs were performed regularly. Results: Muscle function improved in all patients with treatment, and a complete clinical response was achieved in 5 patients. Pulmonary function worsened in 1 patient, who died from respiratory failure, but normalised in 4 patients. Arthropathy progressed despite improvement of myositis and pulmonary status in 2 patients. Discontinuation of treatment was facilitated in 1 patient, although long-term therapy was required in 10 patients. In 2 patients with refractory disease, treatment with intravenous immunoglobulins was successful. Severe side effects of treatment occurred in 7 patients and overall mortality rate was one of 12 (8 %). Conclusion: The antisynthetase syndrome associated with anti-Jo-1-antibodies requires long-term immunosuppressive therapy in most patients. Whereas a complete clinical response of muscular symptoms is frequent, continued deterioration of the pulmonary system may occur despite immunosuppressive treatment, and may lead to fatal outcome. An interdisciplinary therapeutic approach is necessary for best possible results in these patients.     

Celecoxib enhances radiosensitivity in medulloblastoma-derived CD133-positive cells

Objects

Cyclooxygenase-2 (COX-2), the enzyme that converts arachidonic acid to prostaglandins, is overexpressed in a variety of tumors, including medulloblastoma (MB). CD133, a transmembrane glycoprotein, has been suggested as a marker for cancer stem cells in brain tumors. The aim of the present study was to investigate the role of celecoxib, a selective COX-2 inhibitor, in enhancing the effects of ionizing radiotherapy (IR) on medulloblastoma-derived CD133-positive cells (MB-CD133⁺).

Materials and methods

MB-CD133⁺ were isolated from two medulloblastoma cell lines (Daoy and UW228). Then, they were treated with celecoxib in different concentrations, and cell viability was assessed. The assays of cell survival, soft agar, radiosensitivity, colony formation, and apoptotic activity in MB-CD133⁺ treated with celecoxib alone, radiation alone, or celecoxib combined with radiation were further evaluated.

Results

MB-CD133⁺ showed the self-renew ability to form sphere bodies in vitro and regenerate tumors in vivo. The levels of COX-2 mRNA and protein in MB-CD133⁺ were significantly higher than those in MB-CD133^?. The treatment of 30 μM celecoxib could effectively inhibit the abilities of cell proliferation and colony formation and increase IR-induced apoptosis in treated MB-CD133⁺. Furthermore, in vivo study demonstrated that celecoxib significantly enhanced radiosensitivity in MB-CD133⁺-transplanted grafts. Notably, xenotransplantation analysis demonstrated that the treatment of celecoxib could further suppress the expressions of angiogenic and stemnness-related genes in treated MB-CD133⁺ grafts of SCID mice.

Conclusions

Celecoxib presents the potential of radiosensitizing effect in MB-derived cancer stem cells. Therefore, it should be warranted in future trials to enhance the radiotherapeutic effects in MB patients.     

Identity of ED2-positive perivascular cells in rat brain

A controversial, though fundamental, issue in neurobiology concerns the nature, origin, and function of brain macrophages. By immunocytochemical analysis using monoclonal antibodies directed against rat macrophage antigens, i.e., ED1-3, Ox-41, Ox-42, and Ki-M2R, we show that a group of perivascular cells located within the basal membrane of CNS blood vessels are immunoreactive. These cells, which resemble pericytes in terms of their anatomical distribution, are distinct from resting parenchymal microglia immunologically as well as morphologically. Our results demonstrate considerable heterogeneity in the immunophenotype of resident brain macrophages, which may be part of the immune-nervous system interface.     

The decrease of CD8-positive lymphocytes in Alzheimer's disease.

The aim of this study was to analyze the possible association of the changes in systemic immunity in the neurodegenerative diseases and in brain atrophy per se. Therefore we enumerated the numbers and proportions of the CD3-, CD4- and CD8-positive cells and B-lymphocytes in peripheral blood of 136 patients with various neurodegenerative disorders of the brain and 58 healthy age-matched controls. The selective decrease of the CD8-positive lymphocytes was demonstrated in the patients with Alzheimer's disease. In contrast, in the patients with brain atrophy of unknown origin and in the patients with vascular dementia the number of the CD8-positive cells tended to increase. The results suggest that brain degenerative changes are associated with changes in the systemic immunity and the changes are linked with the underlying disorder more than brain degeneration per se.