去甲二氢愈创木酸对人恶性胶质瘤细胞系SHG-44生长和分化的影响

目的探讨去甲二氢愈创木酸（ＮＤＧＡ）对胶质瘤生长和分化的作用及可能机理。方法分别将ＮＤＧＡ加入培养基中和进行单细胞胞浆内显微注射ＮＤＧＡ，观察它对人恶性胶质瘤细胞系ＳＨＧ－４４细胞生长、形态、细胞周期和免疫组化特性的影响。结果经ＮＤＧＡ处理的瘤细胞贴壁率和生长速率受抑制，增殖活性降低，细胞周期也有明显改变；细胞异型性变小，胞浆中胶质细丝增多，且其中ＧＦＡＰ标记增加而ｖｉｍｅｎｔｉｎ标记减少；ｐ５３蛋白和碱性成纤维细胞生长因子（ｂＦＧＦ）表达也降低。单细胞胞浆内注射ＮＤＧＡ（约１．５×１０－１１ｇ／细胞）后上述作用更显著，且作用迅速而持久。结论ＮＤＧＡ对恶性胶质瘤细胞具有抑制生长和诱导分化作用；对血管生成因子表达亦有抑制作用。     

表皮生长因子对胶质瘤干细胞增殖与侵袭的影响

目的研究表皮生长因子(EGF)对胶质瘤干细胞生物学行为影响。方法胰蛋白酶消化法进行胶质瘤细胞原代培养,然后将培养基更换为含有EGF、bFGF、B27的DEME/F12培养基,培养后获得悬浮生长的肿瘤球细胞。悬浮生长的肿瘤球进行免疫荧光及分化实验。CCK-8方法检测EGF对胶质瘤干细胞增殖能力影响。Transwell方法检测EGF对胶质瘤干细胞侵袭能力影响。Western blotting实验研究EGF对胶质瘤干细胞中金属基质蛋白酶(MMP)-2表达影响。结果悬浮生长肿瘤球为胶质瘤干细胞。EGF促进胶质瘤干细胞增殖和侵袭,同时提高胶质瘤干细胞MMP-2表达。结论 EGF促进胶质瘤干细胞的增殖和侵袭与调控MMP-2信号通路相关。     

缺氧增强胶质瘤干细胞“干性”的初步研究

目的研究在体外环境下,缺氧与肿瘤干细胞(CSCs)"干性"的关系。方法选择U87细胞和U251细胞以及原代胶质瘤细胞并进行缺氧处理。用透射电子显微镜法来检测这些细胞的超微结构;用MTT法来检测细胞生长;用流式细胞仪来检测细胞周期以及CD133的表达;用transwell法来检测细胞迁移能力;用集落形成分析来分析细胞的集落形成能力;并用实时定量PCR来检测干细胞以及其分化标志物的m RNA表达。结果缺氧使原代胶质瘤细胞维持在一个未分化的状态,减慢处于相对静止阶段的胶质瘤细胞的生长、增加它们的集落形成能力和胶质瘤细胞的转移,而且能提高干细胞标志物的表达水平。肿瘤干细胞的分化标志物在进行缺氧处理后降低。结论缺氧能够使已分化的胶质瘤细胞"去分化"而使其获得"干性"。     

Twist1对胶质瘤样干细胞的增殖和侵袭影响

目的研究Twist1对胶质瘤样干细胞增殖和侵袭影响。方法通过无血清干细胞培养基方法从原代培养的多形性胶质母细胞瘤中获得悬浮生长的肿瘤细胞球,利用免疫荧光方法及血清诱导分化方法对培养的肿瘤球进行鉴定;通过Western blot方法比较胶质瘤样干细胞和原代培养的多形性胶质母细胞瘤中Twist1表达;SiRNA抑制Twist1表达后,应用CCK-8方法及Transwell方法检测Twist1对胶质瘤样干细胞增殖和侵袭影响。结果无血清干细胞培养基方法可以从原代培养的多形性胶质母细胞瘤中诱导出悬浮生长的肿瘤细胞球;肿瘤细胞球表达肿瘤干细胞标记物CD133和Sox2,在含血清培养基中可以分化为表达神经元和神经胶质细胞标记物的细胞;胶质瘤样干细胞高度表达Twist1;利用siRNA抑制Twist1后,可以抑制胶质瘤样干细胞增殖和侵袭。结论Twist1可能是维持胶质瘤样干细胞干性的关键基因。     

海洋单细胞海藻体外对人脑胶质瘤干细胞生物学活性的影响

背景：海藻具有广阔的药理活性前景,加强其研究对有目的进行应用开发有重要的指导意义。 目的：观察海洋单细胞海藻在体外对人脑胶质瘤干细胞生物学活性的影响。 方法：以酶消化法培养人脑胶质瘤干细胞,流式细胞分选出CD133阳性细胞,细胞传代获得第3代细胞。流式细胞仪检测海洋单细胞海藻作用前后细胞CD133表达变化,免疫组织化学检测贴壁细胞巢蛋白及胶质纤维酸性蛋白的表达。实验组分别加入不同质量浓度的海洋单细胞海藻,阴性对照加不含药的PBS,将稀释成4,6,8,10 g/L的海洋单细胞海藻加入细胞培养液中并作用24,48,72 h,流式细胞仪检测胶质瘤干细胞生长周期,应用酶标仪检测细胞生长抑制情况。 结果与结论：随着浓度和时间的增加,与对照组相比倒置显微镜下可见实验组胶质瘤干细胞不易聚团成球,出现贴壁分化,并逐渐明显;加药后胶质瘤干细胞CD133表达量明显减少;出现的贴壁细胞免疫组织化学染色巢蛋白及胶质纤维酸性蛋白表达阳性;流式细胞仪检测显示,停滞在S、G2/M期细胞数增加,而G0/G1期细胞数减少;随着浓度和时间的增加,胶质瘤干细胞增殖明显抑制,与对照组相比差异有显著性意义(P < 0.05~0.01)。提示海洋单细胞海藻能够抑制人脑胶质瘤干细胞的增殖,并促进其分化,且作用具有浓度和时间依赖性。     

人参皂苷诱导大鼠C6脑胶质瘤细胞的分化

目的:观察人参皂苷对胶质瘤细胞的诱导分化作用,探讨人参皂苷抗胶质瘤作用机制.方法:采用MTT、软琼脂克隆形成率、形态学与神经胶质纤维酸性蛋白(GFAP)免疫荧光等方法鉴定分化程度.结果:10μg/ml人参皂苷即可明显诱导大鼠C6胶质瘤细胞株的细胞分化,表现为增殖抑制,克隆形成能力丧失,细胞突起增长,GFAP表达量升高.结论:一定剂量的人参皂苷可以诱导大鼠脑胶质瘤细胞分化达到抗肿瘤的效果.     

携带绿色荧光蛋白的外源性胶质纤维酸性蛋白基因稳定转染对胶质瘤细胞分化及增殖的影响

目的观察增强胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)基因表达对恶性胶质瘤细胞生物学特性的影响,为胶质瘤诱导分化及基因治疗研究提供理论基础。方法构建携带1．1kb的GFAP cDNA和绿色荧光蛋白(GFP)基因的真核表达载体,采用脂质体转染法将其导入人恶性胶质瘤细胞系SHG-44,G418结合荧光动态监测筛选阳性克隆;采用原位杂交、免疫细胞化学及Western蛋白印迹等方法检测GFAP基因及其蛋白表达;并通过形态学、细胞生长曲线、软琼脂克隆形成及流式细胞分析等观察GFAP基因对胶质瘤细胞形态、增殖和细胞周期的影响。结果转染阳性的SHG-44细胞GFAP mRNA及其蛋白表达增强,瘤细胞形态趋向成熟,突起增多变细,细胞增殖速度减缓,G0／G1期、G2／M期细胞比例降低。结论增强GFAP基因表达可显著抑制胶质瘤细胞增殖并诱导其分化成熟,提示通过基因治疗策略或诱导分化方法上调GFAP基因表达是恶性胶质瘤治疗的新途径。     

恶性胶质瘤细胞SHG-44诱导分化后的差异蛋白质组双向电泳分析

目的用双向电泳分析诺帝诱导胶质瘤细胞SHG-44分化后的差异蛋白质组,为进一步了解这些差异蛋白质的作用打下基础。方法将诺帝诱导胶质瘤细胞SHG-44分化后的总蛋白及其相应的空白对照组细胞总蛋白进行双向电泳分离,重复3次后用PDQuest7.1软件比较分析蛋白质表达差异并获得差异蛋白质的相对分子质量、等电点等信息。结果诺帝诱导胶质瘤细胞SHG-44分化后有23个差异蛋白点,其中21个蛋白点表达下调,2个蛋白点表达上调。结论诺帝诱导胶质瘤细胞SHG-44分化后大部分蛋白质表达下调,推测诺帝诱导胶质瘤细胞SHG-44分化时蛋白表达以抑制作用为主。     

脑胶质瘤中PTEN基因表达的实时定量PCR研究

目的:探讨PTEN基因在胶质瘤中的表达及意义,以及与预后的关系.方法:采用荧光实时定量PCR技术检测44例人脑胶质瘤组织及其19例邻近正常脑组织、7例良性脑肿瘤中的PTEN mRNA及内参GAPDH的表达水平.结果:高分化组(29例)PTEN基因的表达明显高于低分化组(15例)(P＜0.05)这两组分别与良性脑肿瘤组(7例)相比,均有显著差异(P＜0.05).19例胶质瘤与邻近正常脑组织有显著差异(P＜0.000).高分化组和底分化组之间在1年或3年存活率的比较中,存在明显差异(P＜0.05).结论:PTEN基因在胶质瘤的发生及发展中起重要作用,脑胶质瘤中PTEN基因的表达与胶质瘤的恶性程度及预后相关.     

MicroRNA和胶质瘤干细胞

MicroRNA作为近年来发现的调控细胞生长发育的分子,不仅参与生理状态的调控,也参与多种恶性肿瘤的发生发展过程。肿瘤干细胞因其自我更新、多向分化潜能、成瘤性等特点,在肿瘤的发生发展中起着十分重要的作用。本文主要从microRNA的发现与生物学特性、胶质瘤干细胞的发现与生物学标志物以及microRNA对胶质瘤干细胞的作用等方面进行阐述。随着microRNA和胶质瘤干细胞研究的不断进展,将逐渐深入揭示胶质瘤干细胞的生物学特征,为进一步从干细胞角度研究胶质瘤的发病机制及靶向治疗打下基础。     

洛伐他汀对胶质瘤干细胞增殖和凋亡的影响

BACKGROUND:Increasing evidence has shown that lovastatin with less toxicity to normal cells has crucial effects on proliferation, apoptosis and differentiation of various cancer cells. However, its roles in glioma stem cells remain unclear. OBJECTIVE:To explore the effect of lovastatin on proliferation and apoptosis of glioma stem cells. METHODS:Flow cytometric sorting was used to separate glioma stem cells from human glioblastoma cell line U87. Effects of lovastatin on the proliferation and apoptosis of glioma stem cells were determined by MTT and flow cytometry, respectively. Furthermore, expression levels of Ki67, Bax and Bcl-2 in glioma stem cells treated with lovastatin were detected using western blot analysis. RESULTS AND CONCLUSION:The CD133-positive glioma stem cells were sorted from human glioblastoma cell line U87 with a positive percentage of 85%. MTT assay showed that lovastatin inhibited the proliferation of glioma stem cells in dose (5, 10, 20 μmol/L)- and time (24, 48, 72, 96 hours)-dependent manners. Flow cytometry analysis showed that 10 μmol/L lovastatin (48 hours) induced apoptosis in glioma stem cells. In addition, the expression level of Ki67 was decreased by lovastatin treatment in a dose-dependent manner, and the Bcl-2 and Bax expression levels were reduced and increased by 10 μmol/L lovastatin treatment, respectively. In conclusion, lovastatin can inhibit cell proliferation and induce apoptosis of glioma stem cells, and lovastatin may be a potential drug for treatment of brain tumors.     

Platelet-derived growth factor receptor-beta is induced during tumor development and upregulated during tumor progression in endothelial cells in human gliomas.

BACKGROUND: Endothelial cells proliferate during brain development, are quiescent in normal adult brain but proliferate again under pathologic conditions such as glioma growth. The vascular phenotype of low grade glioma is comparable to normal brain, however high grade gliomas are focally highly vascularized and there is associated prominent endothelial cell proliferation. The mechanisms of this change in vascular phenotype are unknown but there is evidence that growth factors play an important role in this process as well as in normal angiogenesis and vascular differentiation. EXPERIMENTAL DESIGN: To investigate whether endothelial cells become activated during tumorigenesis and progression of human gliomas by a platelet-derived growth factor (PDGF) dependent pathway, we analyzed platelet-derived growth factor receptor-beta (PDGFR-beta) expression by in situ hybridization and immunocytochemistry in normal human brain, astrocytoma (grade II), anaplastic oligo-astrocytoma (grade III), and glioblastoma multiforme (grade IV). RESULTS: PDGFR-beta mRNA was not detectable in the vessels of normal human brain, but was expressed in the vasculature of low and high grade gliomas, particularly in endothelial cell proliferations in glioblastomas. The expression of the receptor in the tumor microvessels, was confirmed by double immunofluorescence in which the staining appeared to be in the endothelial cells. Primary cultures of endothelial cells derived from glioblastoma multiforme maintained receptor expression for 2 days in vitro, whereas it was not detectable in vitro in endothelial cells derived from normal brain. Tumor cells in all grades of glioma expressed very little PDGFR-beta mRNA in situ. CONCLUSIONS: Our results indicate that the malignant phenotype in human glial tumors is associated with an upregulation of the PDGFR-beta on endothelial cells of vessels which vascularize the tumor. These findings may contribute to our understanding of the mechanisms that regulate vessel growth and differentiation in normal and pathologic states.     

SHG－44细胞显微注射NDGA诱导分化的初步观察

采用显微注射系统对ＳＨＧ－４４细胞进行单细胞微量注射技术的探讨，并观察去甲二氢愈创木酸（ＮＤＧＡ）注射后的作用。结果显示，体积较小的肿瘤细胞显微注射成功的关键在于注射参数的正确设定、细胞标记、注射针下限值的确定、注射过程中防污染以及操作细致、迅速；ＮＤＧＡ注射ＳＨＧ－４４细胞后的诱导分化作用较其在细胞外培养基中的作用更直接、迅速和显著，亦更持久。     

Human neuroblastoma: Glial induced morphological differentiation

Rat C-6 glioma cells release a factor into culture medium which induces morphological differentiation in the IMR-32 human neuroblastoma cell line. The time course of differentiation and the effects on cell growth differ from those observed in mouse neuroblastoma cells. The effects of other cell lines and the relationship of differentiation to cell growth are discussed.     

MgSO4 and lazaroid (U-83836E) partially protects glioma cells against glutamate toxicity in vitro

In this study, the possible effects of MgSO4 and lazaroid (U-83836E) on glutamate toxicity on glial cells were investigated. C6 and human glioblastoma multiforme cells derived from two patients were grown in an incubator. First, determined IC50 dose of L-glutamate (L-glu) was given for 24 hours and removed, and then respective MgSO4 or U-83836E doses were added to the culture medium. After 24 hours 3-(4,5-Dimethylthyazol-2-yl)-2,5-diphenyltetrazolium bromide, thiazolyl blue (MTT) test was applied. When compared to the L-glu-treated group, MgSO4 at the dose of 0.01 mM induced C6 and human glioma cell growth by 17%, 15% and 5%, respectively. At the dose of 1 microM U-83836E also increased C6 and human glioma cell growth by 12%, 13% and 5%, respectively. In conclusion, although MgSO4 and U-83836E do not strongly block glutamate-induced cell death, it is suggested that reduction of Mg2+ ions and free radical production may have a role in glutamate toxicity on glial cells.     

Glioblastoma stem cells produce vascular endothelial growth factor by activation of a G-protein coupled formylpeptide receptor FPR

Glioma stem cells (GSCs), or stem cell-like glioma cells, isolated from malignant glioma cell lines, were capable of producing vascular endothelial growth factor (VEGF). However, the exact role of such tumour cells in angiogenesis remains unknown. In this study, we isolated a small proportion of CD133+ GSCs from the human glioblastoma cell line U87 and found that these GSCs possessed multipotent differentiation potential and released high levels of VEGF as compared with CD133(-) tumour cells. The CD133+ GSCs also formed larger xenograft tumours that contained higher VEGF immunoreactivity and denser microvessels. Moreover, GSCs expressed a functional G protein-coupled formylpeptide receptor FPR, which was activated by a chemotactic peptide ligand, N-formylmethionyl-leucyl-phenylalanine (fMLF), to mediate calcium flux and the production of VEGF by GSCs. Our results indicate that FPR expressed by human GSCs may play an important role in glioma angiogenesis.     

去甲二氢愈创木酸对胶质瘤细胞诱导的内皮细胞迁移的影响

目的 观察去甲二氢愈创木酸（ＮＤＧＡ）对胶质瘤细胞诱导的内皮细胞迁移的影响。方法 采用微孔滤膜培养小室及室细胞联合培养法,进行人脐静脉内皮细胞系ＥＣＶ－３０４细胞与人恶性胶质瘤细胞系ＳＨＧ－４４细胞的联合培养,并以免疫组化ＳＰ方法检测ＳＨＧ－４４细胞血管内皮生长因子（ＶＥＧＦ）、碱性成纤维细胞生长因子（ｂＦＧＦ）的表达。结果 １００μｍｏｌ／Ｌ的ＮＤＧＡ作用１～３ｄ后,ＳＨＧ－４４细胞ＶＥＧＦ、ｂ     

Impact of static magnetic fields on human myoblast cell cultures

Treatment of skeletal muscle loss due to trauma or tumor ablation therapy still lacks a suitable clinical approach. Creation of functional muscle tissue in vitro using the differentiation potential of human satellite cells (myoblasts) is a promising new research field called tissue engineering. Strong differentiation stimuli, which can induce formation of myofibers after cell expansion, have to be identified and evaluated in order to create sufficient amounts of neo-tissue. The objective of this study was to determine the influence of static magnetic fields (SMF) on human satellite cell cultures as one of the preferred stem cell sources in skeletal muscle tissue engineering. Experiments were performed using human satellite cells with and without SMF stimulation after incubation with a culture medium containing low [differentiation medium (DM)] or high [growth medium (GM)] concentrations of growth factors. Proliferation analysis using the alamarBlue assay revealed no significant influence of SMF on cell division. Real-time RT-PCR of the following marker genes was investigated: myogenic factor 5 (MYF5), myogenic differentiation antigen 1 (MYOD1), myogenin (MYOG), skeletal muscle α1 actin (ACTA1), and embryonic (MYH3), perinatal (MYH8) and adult (MYH1) skeletal muscle myosin heavy chain. We detected an influence on marker gene expression by SMF in terms of a down-regulation of the marker genes in cell cultures treated with SMF and DM, but not in cell cultures treated with SMF and GM. Immunocytochemical investigations using antibodies directed against the differentiation markers confirmed the gene expression results and showed an enhancement of maturation after stimulation with GM and SMF. Additional calculation of the fusion index also revealed an increase in myotube formation in cell cultures treated with SMF and GM. Our findings show that the effect of SMF on the process of differentiation depends on the growth factor concentration in the culture medium in human satellite cultures. SMF alone enhances the maturation of human satellite cells treated with GM, but not satellite cells that were additionally stimulated with serum cessation. Therefore, further investigations are necessary before consideration of SMF for skeletal muscle tissue engineering approaches.     

Reduced glioma growth following dexamethasone or anti-angiopoietin 2 treatment

All patients with glioblastoma, the most aggressive and common form of brain cancer, develop cerebral edema. This complication is routinely treated with dexamethasone, a steroidal anti-inflammatory drug whose effects on brain tumors are not fully understood. Here we show that dexamethasone can reduce glioma growth in mice, even though it depletes infiltrating T cells with potential antitumor activity. More precisely, T cells with helper or cytotoxic function were sensitive to dexamethasone, but not those that were negative for the CD4 and CD8 molecules, including gammadelta and natural killer (NK) T cells. The antineoplastic effect of dexamethasone was indirect, as it did not meaningfully affect the growth and gene expression profile of glioma cells in vitro. In contrast, hundreds of dexamethasone-modulated genes, notably angiopoietin 2 (Angpt2), were identified in cultured cerebral endothelial cells by microarray analysis. The ability of dexamethasone to attenuate Angpt2 expression was confirmed in vitro and in vivo. Selective neutralization of Angpt2 using a peptide-Fc fusion protein reduced glioma growth and vascular enlargement to a greater extent than dexamethasone, without affecting T cell infiltration. In conclusion, this study suggests a mechanism by which dexamethasone can slow glioma growth, providing a new therapeutic target for malignant brain tumors.