人恶性胶质瘤细胞裸鼠脑原位移植瘤FPR表达与VEGF关系

目的观测甲酰肽受体(formylpeptide receptor,FPR)在人恶性胶质瘤细胞系U87细胞裸鼠脑原位移植瘤组织中的表达,以及与血管内皮生长因子(VEGF)的关系,探讨FPR在恶性胶质瘤血管生成过程中的作用。方法培养U87细胞,以立体定向注射技术制作U87裸鼠脑原位移植瘤模型;采用间接免疫荧光染色在激光共聚焦显微镜下分别观测FPR在U87细胞和移植瘤组织的表达;免疫组化方法检测VEGF在U87细胞裸鼠脑移植瘤上的表达。结果培养的U87细胞及其原位移植瘤组织均可见FPR表达,定位于瘤细胞胞膜,VEGF阳性着色主要位于胞质,两者阳性表达程度呈正相关性。结论人恶性胶质瘤细胞系U87细胞裸鼠脑原位移植瘤组织存在FPR表达,与VEGF的产生密切相关,在胶质瘤血管生成过程中可能起重要作用。     

人脑星形细胞肿瘤IL-8的表达及其与血管生成的关系

目的：探讨IL-8,VEGF在脑胶质瘤中的表达及与血管生成的关系。方法：应用由45例人脑星形细胞肿瘤和6例正常脑组织组成的组织芯片，采用免疫组织化学技术分别进行IL-8，VEGF，CD34标记并进行半定量，观测在不同病理分级胶质瘤中的表达及与微血管密度（MVD）之间的关系。结果：6例正常脑组织中不表达IL-8，VEGF。45例脑胶质瘤中，27例IL-8呈阳性表达，32例VEGF呈阳性表达，Ⅲ，Ⅳ级脑胶质瘤中IL-8，VEGF的表达比Ⅰ级、Ⅱ级明显增强，IL-8，VEGF评分为强阳性的胶质瘤内MVD明显高于评分为阴性和阳性的MVD（P<0.01）。IL-8表达与MVD呈正相关（rs=0.64，P<0.01）。VEGF表达与MVD之间呈正相关（rs=0.44，P<0.01）。IL-8表达与VEGF表达之间亦呈正相关（rs=0.56，P<0.01）。结论：IL-8，VEGF的表达与胶质瘤病理分级、MVD密切相关，二者在胶质瘤的血管生成中可能相互关联、共同调节肿瘤血管生成。     

血清剥夺法分离U87细胞系中的胶质瘤干细胞

背景:细胞周期分析已证实,90%以上的干细胞处于G0静止状态,因此采用不含任何生长因子和其他相关营养添加剂的血清剥夺培养基更适合筛选分离肿瘤干细胞。目的:应用血清剥夺法培养胶质瘤U87细胞,并筛选鉴定该细胞系中的胶质瘤干细胞。方法:将U87细胞培养在只含有DMEM和L-谷氨酰胺的培养基中培养6d,筛选出胶质瘤干细胞,接着更换为神经干细胞培养基,观察细胞肿瘤球的形成过程;将肿瘤球接种于血清培养基,观察其在体外的分化特点;免疫荧光鉴定血清剥夺后尚存的细胞、增殖形成的肿瘤球细胞和分化细胞。结果与结论:应用血清剥夺的方法成功地筛选出了表达CD133的肿瘤干细胞,并能增殖形成肿瘤球;肿瘤球可多向分化,子代细胞表达胶质纤维酸性蛋白和神经元特异性烯醇化酶。说明U87细胞系中存在具有自我更新及多向分化能力的胶质瘤干细胞。     

miR-486对胶质瘤干细胞的抑制作用

背景:既往研究发现,mi R-486在胶质瘤干细胞(CD133+)中的表达水平显著低于其在胶质瘤非干细胞(CD133-)中的表达水平,但是mi R-486对CD133+细胞的影响尚不明确。目的:探索mi R-486对CD133+细胞的作用。方法:利用流式细胞分选将U87胶质瘤细胞中分为CD133+和CD133-细胞。通过脂质体转染构建miR-486过表达的胶质瘤干细胞。结果与结论:流式细胞分选和纯化获得高比率的CD133+胶质瘤干细胞。实时反转录PCR检测发现mi R-486在CD133+胶质瘤干细胞的表达水平比CD133-胶质瘤细胞明显下降。脂质体转染成功构建mi R-486过表达的胶质瘤干细胞,体外实验发现mi R-486高表达抑制胶质瘤干细胞的增殖,将其阻滞于G1/S期,并促进凋亡。提示mi R-486对胶质瘤干细胞具有抑制作用。     

U87细胞来源的脑胶质瘤干细胞样细胞B7-H6表达上调且与其生物学特性相关

目的探讨B7家族分子B7-H6在脑胶质瘤干细胞样细胞(GSLC)中的表达及其意义。方法使用亚克隆的方法并利用GSLC在体外形成神经细胞球的特性,从U87细胞株中筛选得到脑GSLC;通过实时定量PCR和流式细胞术检测干细胞标志分子c-myc、性别决定区基因Y盒2(Sox2)、CD133、神经上皮干细胞蛋白(nestin)、CXC趋化因子受体4(CXCR4)的表达;通过化疗药物多柔比星、顺铂、卡铂杀伤鉴定其是否具有耐药特性;通过实时定量PCR及流式细胞术获得脑GSLC中B7家族的表达;采用小干涉RNA(si RNA)干扰下调B7-H6表达,CCK-8法检测细胞增殖的变化。结果使用亚克隆方法分离获得的脑GSLC能在体外形成神经细胞球,上调表达多种干细胞标志分子CD133、nestin、CXCR4,具有显著的耐药特性。B7家族分子B7-H1、B7-H3、B7-H4和B7-H6在得到脑GSLC中均表达,与原代U87细胞相比,细胞膜上的B7-H6的表达发生显著上调,使用si RNA干扰B7-H6表达后,细胞的增殖受到抑制,且c-myc基因的表达也下调。结论 U87细胞来源的脑GSLCB7-H6表达上调,并且与GSLC生物学特性相关。     

血小板因子4基因转染对肺癌细胞中血管生成因子基因表达的影响

目的：进一步探讨人血小板因子4（hPF4）抑制血管生成的作用机制,方法：构建含全长hPF4cDNA重组真核表达载体pcDNA3-hPF4,北朝鲜其转染到有肺巨细胞癌细胞系PLA801D细胞内,应用RT-PCR及免疫组化法观察肿瘤细胞自分泌的血管内皮生长因子（VEGF）、碱性成纤维细胞生长因子（bFGF)、白细胞介素8（IL-8）等的mRNA和蛋白表达水平的变化,结果：导入pcDNA3-hPF4,PLA801D细胞能稳定表达hPF4mRNA,其VEGF、bFGF、IL-8的mRNA和蛋白表达水平均明显降低,hPF4可直接调控VEGF、bFGF、IL-8等基因转录,影响其蛋白质生物合成,结论：提示hPF4可直接下调肿瘤细胞自分泌的VEGF、bFGF及IL-8等血管生成因子基因转录,抑制肿瘤细胞释放肿瘤血管生成因子,是hPF4抗血管生成抑制肿瘤细胞生长的机制之一。     

xCT和促血管生成因子在胶质瘤组织中肿瘤相关巨噬细胞的表达及意义

目的：探讨xCT和促血管生成因子在胶质瘤组织中肿瘤相关巨噬细胞（TAMs）中的表达，以及TAMs对胶质瘤进展的影响。方法：分离并培养人新鲜胶质瘤和正常脑组织中的TAMs。采用荧光实时定量（RT-PCR）技术检测M2表型标记物CD14和CD86,促血管生成因子Arg-1和CD209，以及 xCT-mRNA表达水平。组织冰冻切片免疫荧光染色检测CD68和xCT在胶质瘤中的表达。结果：xCT和CD68在胶质母细胞瘤(WHO°Ⅳ)中的表达明显高于WHO°Ⅱ胶质瘤和正常脑组织，且xCT和CD68共同表达于小胶质细胞中。xCT mRNA在分化程度差的胶质瘤中表达明显增高。此外，小胶质细胞M2表型标记物表达水平(CD14和CD86)、活化状态的TAMs数目和促血管生成因子(Arg-1和CD209)与肿瘤分级相关。结论：TAMs能够通过xCT过表达，促炎症因子和促血管生成因子促进胶质瘤的发展。肿瘤中M2表型的TAMs募集与脑胶质瘤的不良预后相关。     

人胎儿脑室区神经前体细胞的分离纯化、培养及鉴定

目的建立有效的人胎儿神经前体细胞分离及纯化系统。方法取人自然流产胎儿脑室区(VZ)并制备单细胞悬液,采用免疫磁珠法分离CD133阳性及CD133阴性细胞群,体外培养并比较两者增殖能力。用免疫荧光化学方法检测CD133阳性细胞群中神经上皮干细胞蛋白(nestin)的表达及诱导分化后的多向分化潜能。结果纯化后CD133阳性细胞群体外扩增能力强,在无血清培养体系中能形成神经球并可连续传代,免疫荧光化学法检测表明其nestin抗原阳性,诱导分化后可分化为神经元、星形胶质细胞及少突胶质细胞。而CD133阴性细胞在培养体系中多呈单个分散状态,神经球形成数目与CD133阳性细胞有显著性差异(P<0.05)。结论免疫磁珠法可有效分离人胎儿脑内CD133阳性细胞,且该细胞在体外培养体系中具有神经前体细胞的特征。     

乙酰辅酶A羧化酶1对胶质瘤细胞系U87细胞增殖、迁移和侵袭的影响

目的 探讨乙酰辅酶A羧化酶1（ACC1）对人胶质瘤细胞系U87细胞增殖、迁移及侵袭的作用。 方法 Western blotting检测人胶质瘤细胞系U87、U251及U373中ACC1的表达;构建ACC1过表达质粒载体,将过表达ACC1质粒载体瞬时转染至U87细胞中;Western blotting检测转染后U87细胞中ACC1表达情况;MTT实验检测过表达ACC1对U87细胞增殖的影响;Transwell迁移和侵袭实验分别检测过表达ACC1对U87细胞迁移和侵袭的影响;划痕实验检测过表达ACC1对U87细胞划痕愈合能力的影响;Western blotting检测相关蛋白表达变化。 结果 与人胶质瘤细胞系U251和U373相比,U87细胞中ACC1表达较低;ACC1过表达抑制U87细胞增殖（P<0.01）;ACC1过表达抑制U87细胞迁移、侵袭和划痕愈合能力（P<0.01）;ACC1过表达迁移和侵袭相关蛋白波形蛋白(vimentin)、纤维连接蛋白(fibronectin)和尿激酶型纤溶酶原激活剂(uPA)表达下调（P<0.01）,凋亡抑制蛋白Bcl-2和细胞周期蛋白(cyclin) B、cyclin D表达下调（P<0.01）,p-STAT3蛋白表达下调（P<0.01）,细胞周期蛋白P21表达上调（P<0.01）。 结论 过表达ACC1可能通过抑制STAT3活性,抑制人胶质瘤细胞的增殖、迁移和侵袭。     

miR-874-3p靶向结合SDC1抑制胶质瘤血管生成拟态

目的本研究旨在探讨miR-874-3p调控胶质瘤血管生成拟态的潜在分子机制。方法应用实时定量PCR检测miR-874-3p在胶质瘤细胞系U87细胞中的表达水平;应用双荧光素酶报告基因分析系统检测miR-874-3p和SDC1的结合作用;应用CCK-8法、transwell法、体外管形成实验检测miR-874-3p或SDC1表达变化对U87细胞增殖、迁移、侵袭和管形成能力的影响。结果 miR-874-3p在U87细胞中低表达,过表达miR-874-3p显著抑制U87细胞的增殖、迁移、侵袭和管形成能力。SDC1在U87细胞中的表达上调,沉默SDC1显著抑制U87细胞增殖、迁移、侵袭和管形成能力。miR-874-3p靶向SDC1 3'UTR。结论 miR-874-3p通过靶向结合SDC1抑制胶质瘤血管生成拟态。     

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.     

The chemokine CXCL12 and its receptor CXCR4 promote glioma stem cell-mediated VEGF production and tumour angiogenesis via PI3K/AKT signalling

Chemokines and their receptors are actively involved in inflammation, immune responses, and cancer development. Here we report the detection of CD133(+) glioma stem-like cells (GSCs) co-expressing a chemokine receptor CXCR4 in human primary glioma tissues. These GSCs were located in areas adjacent to tumour vascular capillaries, suggesting an association between GSCs and tumour angiogenesis. To test this hypothesis, we isolated CD133(+) GSCs from surgical specimens of human primary gliomas and glioma cell lines. As compared to CD133(-) cells, CD133(+) GSCs expressed significantly higher levels of CXCR4 mRNA and protein, and migrated more efficiently in response to the CXCR4 ligand CXCL12. In addition, CXCL12 induced vascular endothelial growth factor (VEGF) production by CD133(+) GSCs via activation of the PI3K/AKT signalling pathway. Furthermore, knocking down of CXCR4 using RNA interference or inhibition of CXCR4 function by an antagonist AMD3100 not only reduced VEGF production by CD133(+) GSCs in vitro, but also attenuated the growth and angiogenesis of tumour xenografts in vivo formed by CD133(+) GSCs in SCID mice. These results indicate that CXCL12 and its receptor CXCR4 promote GSC-initiated glioma growth and angiogenesis by stimulating VEGF production.     

猕猴骨髓干细胞动员后外周血干细胞、免疫细胞亚群及细胞因子变化

目的:观察GM-CSF动员猕猴骨髓干细胞后外周血干细胞、免疫细胞亚群和细胞因子含量的动态变化,为临床干细胞动员及用于治疗疾病提供参考依据.方法:健康猕猴连续5 d,皮下注射GM-CSF 8 μg/(kg·d),分别于0、2、4、6、8、10 d采集外周血,血细胞分析仪计数白细胞(WBC)总数、淋巴细胞和中性粒细胞比例,流式细胞术(FCM)测定CD34 、CD133 、CD3 、CD4 、CD8 、CD56 细胞比例,酶联免疫分析法测定血清TNF-α、IL-1β、IL-2含量.结果:WBC、中性粒细胞、CD34 、CD133 细胞数量和比例均同步升高(P＜0.01),到动员第6天时达到高峰,细胞数量分别为正常水平的6.4、9.1、117和163.3倍,其中CD34 、CD133 第8天时恢复正常,而WBC、中性粒细胞仍高于正常水平(P＜0.05).CD3 、CD4 、CD8 、CD56 细胞的数量增加,细胞数在第6天时分别为动员前的4.1、4.0、2.9和4.3倍,但比例下降(P＜0.01),到第6天达到最低(P＜0.001),随后逐渐升高至正常以上水平并持续至第10天(P＜0.05).TNF-α、IL-1β、IL-2浓度于动员后6 d内明显升高(P＜0.01),其中TNF-α、IL-1β浓度至8 d恢复正常(P＞0.05),IL-2浓度升高幅度较大并至少持续至第10天(P＜0.01).结论:连续5 d动员猕猴骨髓干细胞可使外周血中CD34 、CD133 细胞比率短暂升高,WBC、中性粒细胞比例持续升高,使CD3 、CD4 、CD8 、CD56 细胞绝对数增加,TNF-α、IL-1β、IL-2浓度升高,表明GM-CSF动员猕猴骨髓干细胞可在细胞和免疫调节因子水平提高免疫功能.     

Invasion of primary glioma- and cell line-derived spheroids implanted into corticostriatal slice cultures

Gliomas are highly invasive tumors and the pronounced invasive features of gliomas prevent radical surgical resection. In the search for new therapeutics targeting invasive glioma cells, in vivo-like in vitro models are of great interest. We developed and evaluated an in vivo-like in vitro model preserving the invasive features and stem cell features of glioma cells. Fluorescently labelled primary glioma spheroids and U87MG cell line-derived spheroids were implanted into organotypic rat corticostriatal slice cultures and the invasion was followed over time by confocal microscopy. The invasion was validated immunohistochemically with paraffin sections using a human-specific vimentin antibody. Moreover, the preservation of immature stem cell features was evaluated immunohistochemically using the stem cell markers CD133, Sox2, Bmi-1 and nestin. The confocal and immunohistochemical results showed that the primary glioma spheroid area was constant or decreasing after implantation, with a clear increase in the number of invading cells over time. In contrast, the U87MG spheroid area increased after implantation, with no convincing tumor cell invasion. High levels of Bmi-1 and nestin were found in all spheroids, whereas high levels of Sox2 and low to moderate levels of CD133 were only found in the primary spheroids. In conclusion, the invasion of gliomas is preserved using primary glioma spheroids. Some stem cell features are preserved as well, making this model useful in drug development elucidating both invasion and cancer stemness at the early in vitro level.     

胶质瘤细胞及其干细胞的放疗敏感性差异

目的探讨脑胶质瘤细胞与脑肿瘤干细胞(BTSCs)的放射敏感性差异。方法胶质瘤细胞接种于含10%胎牛血清的DMEM/F12培养基进行培养。用血清剥夺法(无血清的DMEM/F12培养基添加bFGF、EGF和B27,即干细胞培养液)培养胶质瘤干细胞。CD133免疫细胞化学染色鉴定BTSCs。用直线加速器分别以0、2、4、6、8、10Gy X线照射胶质瘤细胞及BTSCs,然后继续培养36h。流式细胞仪检测上述细胞中CD133+细胞比率。采用MTT法检测细胞的存活情况。结果胶质瘤细胞中存在BTSCs,后者能在干细胞培养液中存活并悬浮生长,增殖形成克隆球,具有自我更新和增殖能力,表达特异性标志物CD133。胶质瘤细胞及BTSCs在X线照射后,其增殖能力都下降,但BTSCs的下降幅度远没有胶质瘤细胞大(P0.05)。CD133+比率显示,X射线对普通胶质瘤细胞中极其微量的CD133+细胞几乎没有影响,而BTSCs中的CD133+细胞的比例在0-10GyX射线放射过程中,与放射剂量成正比(P0.05)。结论 BTSCs的放射敏感性明显低于胶质瘤细胞,其机制可能与放疗过程中CD133+细胞的增加有关。     

Overexpression of CXC chemokine receptors is required for the superior glioma-tracking property of umbilical cord blood-derived mesenchymal stem cells

Our observations indicate that umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) have a strong migration capacity toward the human glioma cell line, U-87 MG, LN18, U138, and U251, when compared to several other cancer cell lines. In order to identify soluble factors that function to attract UCB-MSCs, we used cytokine antibody arrays to screen changed cytokines in conditioned media from U-87 MG cells. Among these, interleukin-8 (IL-8) and growth-related oncogene (GRO-alpha) enhanced UCB-MSC migration. Furthermore, antibodies treatment against the IL-8 receptors reduced these migration events and overexpression of IL-8 in cells with lower level of IL-8 such as A549 could induce UCB-MSC migration. Since we found that the capacity of UCB-MSC migration is much higher than that of bone marrow-derived MSCs (BM-MSCs) toward either U-87 MG cells or recombinant IL-8, we compared the levels of the IL-8 receptor, CXC chemokine receptor 1 (CXCR1) and CXCR2 between two kinds of MSCs by RT-PCR and immunostaining. Expression levels of two receptors were much higher in UCB-MSCs than in BM-MSCs. These data suggest that higher levels of two IL-8 receptors could influence downstream signaling events affecting superior UCB-MSC migration toward the glioma cells.     

Hypoxia-inducible factor 1 and VEGF upregulate CXCR4 in glioblastoma: implications for angiogenesis and glioma cell invasion

Hypoxia and hypoxia-inducible factor-1 (HIF-1) play a critical role in glioblastoma multiforme (GBMs). CXCR4 is involved in angiogenesis and is upregulated by HIF-1alpha. CXCR4 is a chemokine receptor for stromal cell-derived factor-1 (SDF-1)alpha, also known as CXCL12. We hypothesized that CXCR4 would be upregulated by hypoxia in GBMs. First, we investigated the expression of HIF-1alpha and CXCR4 in GBMs. CXCR4 was consistently found colocalized with HIF-1alpha expression in pseudopalisading glioma cells around areas of necrosis. In addition, angiogenic tumor vessels were strongly positive for CXCR4. Next, we tested the in vitro effect of hypoxia and vascular endothelial growth factor (VEGF) on the expression of CXCR4 in glioma cell lines and in human brain microvascular endothelial cells (HBMECs). Exposure to hypoxia induced significant expression of CXCR4 and HIF-1alpha in glioma cells, whereas treatment with exogenous VEGF increased CXCR4 expression in HBMECs. We also transfected U87MG glioma cells with an HIF-1alpha construct and observed that CXCR4 was upregulated in these cells even in normoxic conditions. We then used a lentivirus-mediated shRNA expression vector directed against HIF-1alpha. When exposed to hypoxia, infected cells failed to show HIF-1alpha and CXCR4 upregulation. We performed migration assays under normoxic and hypoxic conditions in the presence or absence of AMD3100, a CXCR4 inhibitor. There was a significant increase in the migration of U87MG and LN308 glioma cells in hypoxic conditions, which was inhibited in the presence of AMD3100. These studies demonstrate the critical role played by hypoxia and CXCR4 in glioma cell migration. Based on these studies, we suggest that hypoxia regulates CXCR4 in GBMs at two levels. First, through HIF-1alpha in the pseudopalisading tumor cells themselves and, secondly, by the VEGF-stimulated angiogenic response in HBMECs. We believe this knowledge may lead to a potentially important two-pronged therapy against GBM progression using chemotherapy targeting CXCR4.     

胃癌单克隆细胞球和CD44＋及CD133＋亚群成瘤能力的比较

目的研究胃癌干细胞亚群的筛选方法,探讨建立胃癌干细胞稳定亚群的可行性。方法利用流式细胞仪从MKN45、MKN25、SGC7901细胞株和人胃癌组织中分选出不同CD44和CD133表型的亚群,比较不同亚群和无血清培养基培养的单克隆细胞球细胞在裸鼠移植瘤模型中的成瘤能力。结果不同细胞的CD44＋和CD133＋亚群在低数量级时几乎不具有裸鼠皮下成瘤能力,在高数量级接种时成瘤能力与母系细胞差异无统计学意义;单克隆细胞球细胞在各数量级下的成瘤能力、瘤体积和生瘤速度均显著高于母系细胞。结论与CD44和CD133等细胞表面标志物筛选法相比,单克隆细胞球细胞可更好的作为胃癌干细胞研究的细胞学基础。     

The Ultrastructural Difference between CD133-positive U251 Glioma Stem Cells and Normal U251 Glioma Cells

Glioma stem cells (GSC) have higher tumorigenic potential and stronger chemoresistance and radioresistance than normal glioma cells. The mechanisms behind these phenomena have remained elusive. The authors have isolated CD133-positive U251 GSCs from U251 glioma cells and detected the expression of stem cell markers (CD133 and nestin) of U251 GSCs by immunofluorescence staining. Then the ultrastructures of U251 GSCs and normal U251 glioma cells were observed by transmission electron microscopy and the ultrastructural differences between them were compared. Increased cell nucleus atypia, rougher endoplasmic reticulum, and more microvilli were observed in CD133-positive U251 GSCs than in normal U251 glioma cells. In summary, these ultrastructural differences support the hypothesis that GSCs have stronger tumorigenic ability and resistance to chemotherapy and radiotherapy.