Effects of Raf-1 siRNA on human cerebral microvascular endothelial cells: a potential therapeutic strategy for inhibition of tumor angiogenesis

The serine/threonine kinase Raf-1 is involved in the regulation of tumor cell survival, proliferation and metastasis formation, and has therefore emerged as a promising target for cancer therapy. In addition, Raf-1 activity mediates proliferation of endothelial cells thereby promoting angiogenesis and invasive growth of various tumors, including highly vascularized malignant glioblastoma. The aim of this study was to evaluate the effects of small inhibitory RNA (siRNA) directed against Raf-1 on viability, proliferation and motility in glioma cells and cerebral endothelial cells. Half-quantitative RT-PCR and Western blotting revealed efficient siRNA-mediated Raf-1 down regulation in glioma cells (U373, U251) and in human cerebral microvascular endothelial cells (HCMEC). Surprisingly, Raf-1 gene silencing failed to affect cell survival, proliferation or migration activity in the glioblastoma cell lines. In HCMEC, however, pronounced decrease of cell survival and significant inhibition of tube formation was achieved by Raf-1 siRNA compared to non-functional siRNA or vehicle controls. In conclusion, Raf-1 silencing appears as a potential therapeutic strategy to inhibit brain tumor angiogenesis and thereby outgrowth of highly vascularized glioblastoma multiforme, whereas direct cytotoxic effects of Raf-1 knockdown in tumor cells may vary.     

Notch1信号系统在氟西汀上调大鼠海马神经再生中的作用

目的 了解慢性氟西汀干预正常大鼠所导致的海马神经再生上调与Notch1信号系统功能改变的关系.方法 应用大鼠腹腔注射氟西汀建立在体模型,分为对照组、14 d干预组、28 d干预组(n=12),采用免疫组化、real time PCR和Western blot,测定大鼠海马神经干细胞的增殖、存活和分化以及Notch1信号通路各个因子(NICD、Hes1、Hes5、Jag1)的基因及蛋白表达水平的改变.结果 ①与对照组(2919.50±188.80)比较,14 d氟西汀干预组(3706.50±228.04)、28 d氟西汀干预组(4334.33±217.48)海马齿状回神经干细胞增殖明显增加(P<0.001);与对照组(2404.50±148.77)相比,Flu干预28 d组(3273.16±156.68)海马齿状回神经干细胞存活明显增加(P<0.001);与对照组比较,氟西汀干预组NeuN/BrdU、GFAP/BrdU比例无明显差异(P＞0.05).②与对照组[NICDmRNA (0.30±0.03),Hes1mRNA (0.53±0.03),Hes5mRNA (0.21±0.02),Jag1mRNA(1.04±0.07)]比较,氟西汀(Flu)干预14d组[NICDmRNA (0.45±0.05),Hes1mRNA (0.65±0.06),Hes5mRNA (0.31±0.06),Jag1mRNA(2.46±0.39)]和Flu干预28 d组[NICDmRNA (0.42±0.03),Hes1mRNA (0.85±0.06),Hes5mRNA (0.39±0.02),Jag1mRNA(3.21±0.34)]Notch1信号通路各因子基因水平均明显升高(P<0.01或P<0.001).③与对照组[NICD(2.36±0.17),Hes1(1.09±0.25),Jag1(2.33±0.31)]比较,Flu干预14 d组[NICD(3.20±0.25),Jag1(2.86±0.25)]和Flu干预28 d组[NICD(3.40±0.19),Hes1(1.43±0.13),Jag1(3.35±0.14)]NICD、Hes1、Jag1蛋白水平明显升高,差异有统计学意义(P<0.01或P<0.001).与对照组Hes5比较,Flu干预14 d组Hes5和Flu干预28 d Hes5蛋白水平无改变,差异无统计学意义(P＞0.05).结论 氟西汀促进大鼠海马齿状回神经干细胞的增殖和存活,但对分化无影响;同时,海马Notch信号功能激活,提示Notch1信号系统可能参与氟西汀介导的大鼠海马神经再生上调.     

Jagged1 regulates the activation of astrocytes via modulation of NFκB and JAK/STAT/SOCS pathways

The Notch pathway is implicated in many aspects of the central nervous system (CNS) development and functions. Recently, we and others identified the Notch pathway to be involved in inflammatory events of the CNS. To understand the implication of this pathway on astrocytes, we have studied the Jagged‐Notch‐Hes pathway under inflammatory conditions. LPS exposure induced an upregulation of Jagged1 expression on cultured astrocytes. To address the role of Jagged1 in the modulation of inflammation, we used a siRNA mediated silencing of Jagged1 (siRNA J1). Jagged1 inhibition induced important variations on the Notch pathway components like Hes1, Hes5, Notch3, and RBP‐Jκ. siRNA J1 repressed the mRNA expression of genes known as hallmarks of the gliosis like GFAP and endothelin(B) receptor. On activated astrocytes, the inhibition of Jagged1 had antiinflammatory effects and resulted in a decrease of LPS‐induced proinflammatory cytokines (IL1β, IL1α, and TNFα) as well as the iNOS expression. The inhibition of Jagged1 induced a modulation of the JAK/STAT/SOCS signaling pathway. Most interestingly, the siRNA J1 decreased the LPS‐induced translocation of NFκB p65 and this could be correlated to the phosphorylation of IκBα. These results suggest that during inflammatory and gliotic events of the CNS, Jagged1/Notch signaling sustains the inflammation mainly through NFκB and in part through JAK/STAT/SOCS signaling pathways. © 2009 Wiley‐Liss, Inc.     

Role of vincristine in the inhibition of angiogenesis in glioblastoma

Objective: Vincristine, a microtubule-destabilizing drug, was found to exhibit anti-angiogenic effects and anti-tumoral activity. However, the precise mechanism by which vincristine inhibits angiogenesis in glioblastomas is not well understood. Our aim was to investigate whether vincristine affects vascular endothelial growth factor (VEGF) expression in glioblastoma cells and determine whether it is mediated by the downregulation of hypoxia-inducible factor-1α (HIF-1α).

Methods: We investigated the expression of HIF-1α in glioblastoma tissues resected from patients and in human glioblastoma cell lines using immunohistochemistry, Western blot analysis, and immunocytochemistry. In addition to an MTT assay assessing the effect of vincristine on cell proliferation and viability, the effects of vincristine on VEGF mRNA expression and HIF-1α protein were examined using real-time RT-PCR and Western blot analysis under 1% O₂ (hypoxia).

Results: HIF-1α was expressed in the majority of glioblastoma tissues and was detected mainly in the nucleus. Strong immunoreactivity for HIF- 1 α was found often in the hypercellular zones. Under hypoxic conditions, HIF-1α protein levels in the glioblastoma cell lines increased, primarily localizing into the nucleus similar to glioblastoma tissues. Exposure of glioblastoma cells to vincristine resulted in enrichment of the G₂-M fraction of the cell cycle, which suggests that vincristine-mediated growth inhibition of glioblastoma is correlated with mitotic inhibition. Using doses lower than those found to reduce the viability and proliferation of cells by 50% (IC₅₀), vincristine decreased both the expression of VEGF mRNA and the level of HIF-1α protein in hypoxic glioblastoma cells. In addition, following exposure to vincristine, the expression of VEGF mRNA was correlated with HIF-1α protein levels.

Conclusions: Our results suggest that the mechanism by which vincristine elicits an anti-angiogenic effect in glioblastomas under hypoxic conditions might be mediated, in part, by HIF-1α inhibition.     

下调Notch-1基因表达抑制胶质母细胞瘤的增殖活性研究

目的 应用siRNA下调胶质母细胞瘤TJ-905细胞株Notch-1基因抑制肿瘤增殖活性.方法 实验设以Notch-1为基因靶点的siRNA1、siRNA2、siRNA3转染组、阴性对照组和空白对照组,采用OligofectamineTM2000二次转染方法将 siRNA转移到细胞内.实时荧光定量PCR检测siRNA对Notch-1基因表达的抑制作用,筛选出最优siRNA序列用于后续研究;四唑盐(MTT)比色法检测细胞增殖活性;使用Notch-1 siRNA或阴性siRNA转染的TJ-905细胞悬液注射裸鼠腋下,观察移植瘤生长情况,比较肿瘤体积,观察3周后处死动物,切片行HE染色鉴定.结果 (Ⅰ)siRNA明显抑制Notch-1 mRNA的表达水平,空白对照组、阴性对照组、siRNA1、2、3转染组Notch-1 mRNA 表达率分别为1.00±0.07、1.04±0.05、0.11±0.02、0.12±0.01、0.77±0.03,siRNA1为最优序列.(2)siRNA1转染组细胞增殖活性显著降低.(3)接种Notch-1 siRNA1转染的TJ-905细胞裸鼠肿瘤生长速度明显低于接种阴性转染的TJ-905细胞裸鼠,裸鼠处死后观察肿瘤体积:Notch-1 siRNA1转染组为(748.3±154.3)mm3,阴性转染组为(1739.2±249.7)mm3,病理切片HE染色证明为多形性胶质母细胞瘤.结论 化学合成的靶向Notch-1基因的siRNA可以显著地下调Notch-1基因表达水平,抑制肿瘤增殖活性.为进一步进行胶质瘤的基因治疗研究奠定基础.

Abstract：

Objective To study the inhibitory effect of siRNA on glioblastoma (GBM) Notch-1 gene expression in addition to the growth of TJ- 905 glioblastoma.Methods Three Small interference RNAs (siRNAs) targeting Notch- 1 gene named siRNA1,siRNA2,siRNA3 were synthesized chemically in vitro with gene bank BLAST.TJ-905 cells were transfectedtwice with the siRNA by using OligofectamineTM2000.The nontransfected cells and nonspecific siRNAs transfected cells were taken as blank and nonsense controls.Down - regulation of Notch- 1 was demonstrated by real- time RT- PCR,according to the result of QRT- PCR we therefore selected the most effective siRNA for further study.Cell proliferation was measured by MTT analysis.Male Balb/C nude mice were injected subcutaneously with Notch- 1 siRNA- or nonsense siRNA- transfected TJ-905 cells,tumor sizes were measured every 4 days.HE stain was used to determine the property of tumor.Resuts(1) The expression of Notch- 1 mRNA in blank,nonsense controls,siRNA1,2,3 groups were 1.00±0.07,1.04±0.05,0.11 ±0.02,0.12 ±0.01,0.77 ±0.03 by real-time RT- PCR scan analysis,the most effective siRNA was siRNA1.(2) An inhibitory proliferation and growth can be induced in siRNA1 transfected group.(3)Nude mice xenografted with cell suspensions from the nonsense siRNA group developed tumors with a significantly increased volume (from the 18th days) as compared to mice that received the Notch- 1 siRNA1- treated cells.The final tumor volume were less in nude mice injected with Notch- 1 siRNA (748.3 ±154.3 )mm3 compared to nonsense si RNA injection (1739.2 ± 249.7 )mm3,HE stain demonstrate that tumor was multiformity glioblastoma.Conclusion The chemically synthesized siRNA targeted Notch- 1 gene could down -regulate the expression of Notch- 1.In addition,an inhibitory proliferation and growth were induced when compared with the control cells in vitro and in vivo.It was suggested that the suppression of Notch- 1 expression and the inhibition of growth provide a new way to glioma gene therapy.     

RNAi抑制STAT3对胶质瘤干细胞的生长抑制作用

目的 建STAT3的RNAi载体干扰STAT3的表达,在体内外研究对胶质瘤干细胞的影响。方法 构建STAT3基因shRNA的腺病毒表达载体,感染人胶质瘤干细胞。采用RT -PCR、Western blot检测STAT3基因的干扰效果,用MTT和流式细胞仪检测干细胞的增殖、凋亡,并观察对荷瘤裸鼠的影响。结果转染重组腺病毒Ad - STAT3 siRNA后,胶质瘤干细胞的STAT3的mRNA表达率为(41.5±7.3)％,蛋白表达率为（31.2±6.4）％,细胞的增殖受到抑制,凋亡率为(23.8±6.1)％,同时荷瘤裸鼠的肿瘤生长受到抑制,生存期延长。结论重组腺病毒介导的STAT3 RNAi可显著抑制靶基因的表达与活化,显著抑制胶质瘤干细胞的生长功能,为基于胶质瘤干细胞的功能治疗提供了基础理论。     

小分子干扰RNA沉默GLI1基因抑制U87胶质瘤细胞的增殖

目的通过研究小分干扰RNA(siRNA)沉默胶质瘤相关癌基因1(GLI1)后对U87胶质瘤细胞的细胞增殖的影响。 方法通过合成并转染siRNA抑制U87细胞内GLI1的表达,并记为GLI1 siRNA组,转染无意义的siRNA和未转染siRNA的细胞作为对照组。采用MTT法分析各组U87细胞的增殖,采用流式细胞技术分析各组细胞周期变化。 结果Western blotting结果显示,与对照组和空白对照组相比,实验组U87细胞内GLI1的表达量显著下降(P<0.05);MTT实验发现,U87细胞的活性与阴性对照组和空白对照组相比显著下降,并且随着培养时间的延长,细胞的活性下降程度越明显(P<0.05);通过流式细胞术分析细胞周期显示,与对照组相比,实验组U87细胞的细胞周期被阻滞于G1期,并且其S期细胞的数量明显下降(P<0.05)。 结论抑制GLI1的表达可有效抑制U87胶质瘤细胞的增殖,提示GLI1可作为胶质瘤的一种潜在治疗靶点。     

Divergent effects of oncostatin M on astroglioma cells: influence on cell proliferation, invasion, and expression of matrix metalloproteinases

Oncostatin M (OSM), a cytokine of the interleukin-6 (IL-6) family, can either promote or inhibit cell growth in various normal and tumor cells. We addressed the effects of exogenous OSM on the proliferation and invasion of human astroglioma cells. In addition, we investigated one of the possible mechanisms involved: modulation of matrix metalloproteinase (MMP) expression and enzymatic activity. We found that OSM inhibited the proliferation of two human astroglioma cell lines (CH235-MG and U87-MG), and that this effect was not due to apoptosis. The inhibitory effect of OSM on proliferation was mediated through the gp130/OSMRbeta receptor complex. To extend these findings, we analyzed the effects of OSM on primary tumor cells from glioblastoma patients. OSM suppressed the proliferation of primary glioblastoma cells, but not that of normal astrocytes. Interestingly, OSM did not suppress astroglioma cell invasion. This may be due to the differential regulation of MMPs by OSM. We found that OSM inhibited the constitutive expression of MMP-2, while MMP-9 expression was enhanced in astroglioma cell lines. We conclude that OSM inhibits proliferation of human astroglioma cells and primary glioblastoma cells via the gp130/OSMRbeta receptor complex. However, OSM does not affect the invasive capacity of the astroglioma cells, which may be due to the divergent effects of OSM on MMP-2 and MMP-9 expression. Collectively, these findings suggest a complex role for OSM in astroglioma biology.     

Notch activity is downregulated just prior to retinal ganglion cell differentiation

The Notch signaling pathway is important at several stages of retinal development including the differentiation of retinal ganglion cells and Muller glia. The downstream effectors of Notch signaling, Hes1 and Hes5, have been shown to be critical in the retina as well. While Notch activity directly regulates Hes1 and Hes5 elsewhere in the nervous system, it has been unclear whether Hes1 and/or Hes5 are directly regulated by Notch activity in the developing retina. Here, we report that both Hes1 and Hes5 are directly regulated by Notch activity during retinal development. Using fluorescence-based Hes1 and Hes5 reporter constructs, we can monitor Notch activity in progenitor cells in the intact retina, and we find that Notch activity is downregulated just prior to retinal ganglion cell differentiation.     

Enhanced cell growth and tumorigenicity of rat glioma cells by stable expression of human CD133 through multiple molecular actions

CD133 (Prominin‐1/AC133) is generally treated as a cell surface marker found on multipotent stem cells and tumor stem‐like cells, and its biological function remains debated. Genetically modified rat glioma cell lines were generated by lentiviral gene delivery of human CD133 into rat C6 glioma cells (hCD133⁺‐C6) or by infection of C6 cells with control lentivirus (mock‐C6). Stable hCD133 expression promoted the self‐renewal ability of C6‐formed spheres with an increase in the expression of the stemness markers, Bmi‐1 and SOX2. Akt phosphorylation, Notch‐1 activation, and Notch‐1 target gene expression (Hes‐1, Hey1 and Hey2) were increased in hCD133⁺‐C6 when compared to mock‐C6. The inhibition of Akt phosphorylation, Notch‐1 activation, and Hes‐1 in hCD133⁺‐C6 cells effectively suppressed their clonogenic ability, indicating that these factors are involved in expanding the growth of hCD133⁺‐C6. An elevated expression of GTPase‐activating protein 27 (Arhgap27) was detected in hCD133⁺‐C6. A decline in the invasion of hCD133⁺‐C6 by knockdown of Arhgap27 expression indicated the critical role of Arhgap27 in promoting cell migration of hCD133⁺‐C6. In vivo study further showed that hCD133⁺‐C6 formed aggressive tumors in vivo compared to mock‐C6. Exposure of hCD133⁺‐C6 to arsenic trioxide not only reduced Akt phosphorylation, Notch‐1 activation and Hes‐1 expression in vitro, but also inhibited their tumorigenicity in vivo. The results show that C6 glioma cells with stable hCD133 expression enhanced their stemness properties with increased Notch‐1/Hes‐1 signaling, Akt activation, and Arhgap27 action, which contribute to increased cell proliferation and migration of hCD133⁺‐C6 in vitro, as well as progressive tumor formation in vivo.     

Effect of neurosphere size on the growth rate of human neural stem/progenitor cells

Neural stem/progenitor cells (NSPCs) proliferate as aggregates in vitro, but the mechanism of aggregation is not fully understood. Here, we report that aggregation promotes the proliferation of NSPCs. We found that the proliferation rate was linear and depended on the size of the aggregate; that is, the population doubling time of the NSPCs gradually decreased as the diameter approached 250 micro m and flattened to a nearly constant value beyond this diameter. Given this finding, and with the intent of enhancing the efficiency of human NSPC expansion, we induced the NSPCs to form aggregates close to 250 micro m in diameter quickly by culturing them in plates with U-bottomed wells. The NSPCs formed aggregates effectively in the U-bottomed wells, with cell numbers approximately 1.5 times greater than those in the aggregates that formed spontaneously in flat-bottomed wells. In addition, this effect of aggregation involved cell-cell signaling molecules of the Notch1 pathway. In the U-bottomed wells, Hes1 and Hes5, which are target genes of the Notch signal, were expressed at higher levels than in the control, flat-bottomed wells. The amount of cleaved Notch1 was also higher in the cells cultured in the U-bottomed wells. The addition of gamma-secretase inhibitor, which blocks Notch signaling, suppressed cell proliferation in the U-bottomed wells. These results suggest that the three-dimensional architecture of NSPC aggregates would create a microenvironment that promotes the proliferation of human NSPCs.     

Notch1信号系统与抑郁模型大鼠海马神经重塑障碍

目的 探讨Notch1信号系统在抑郁海马神经再生障碍中的作用.方法 选择行为学评分相近的68只Sprague-Dawley大鼠,分为对照组、对照+氟西汀组、慢性不可预知温和应激(CUMS)组,CUMS+氟西汀组,每组17只.应用CUMS建立抑郁模型后进行行为学评估;采用免疫组织化学方法检测大鼠海马神经干细胞的增殖和存活;采用实时定量聚合酶链反应和蛋白免疫印迹方法测定Notchl信号通路各个因子(NICD、Hes1、Hes5、Jag1)的基因及蛋白表达水平的改变.结果 (1)干预前,各组体质量、糖水偏好、旷场试验、强迫游泳评分的差异均无统计学意义(P>0.05);干预后,与对照组比较,CUMS组糖水偏好、水平得分和垂直得分降低,漂浮不动时间增加,差异均有统计学意义(P<0.001);与CUMS组比较,CUMS+氟西汀组糖水偏好、水平得分和垂直得分增加,漂浮不动时间降低,差异均有统计学意义(P<0.01);(2)神经干细胞的增殖和存活:与CUMS组(1900.33±104.10)比较,CUMS+氟西汀组(3047.61±158.29)神经干细胞的增殖数显著上升,差异有统计学意义(P<0.01);与CUMS组(1845.33±126.88)比较,CUMS+氟西汀组(2704.21±154.31)神经干细胞的存活数显著上升,差异有统计学意义(P<0.01);(3)海马Notch1信号通路基因和蛋白的表达:CUMS+氟西汀组小鼠抗大鼠Notch1(NICD)mRNA、Hes1 mRNA、Hes5 mRNA、Jag1 mRNA基因表达与CUMS组比较显著上升,差异均有统计学意义(P<0.01);CUMS+氟西汀组NICD、Hes5、Jag1蛋白水平与CUMS组比较显著上升,差异均有统计学意义(P<0.01).结论 Notch1信号系统可能参与慢性应激模型大鼠海马神经再生障碍;氟西汀可能通过上调Notch1信号系统改善海马神经再生,从而缓解大鼠抑郁症状.

Abstract：

Objective To investigate whether the effect of fluoxetine on hippocampal neurogenesis involves Notch1 signaling after chronic stress. Methods Sixty-eight male Sprague-Dawley rats were divided into control group, control + fluoxetine group, depression model group and depression model + fluoxetine group. Chronic unpredictable mild stress (CUMS) was used to make up depression animal model. The function of Notch1 signaling was measured by real-time PCR and western blotting. Simultaneously,hippocampal neurogenesis was monitored by assessing cell proliferation and survival. Results (1) Before starting CUMS protocol, the animals exhibited equivalent weight, sucrose preference, number of squares crossed, number of rearing, and immobility time in behavioral test. Twenty-eight days after CUMS protocol,these parameters were significantly difference in rats exposed to CUMS compared with the controls (sucrose preference, number of squares crossed, number of grooming and rearing, and immobility time, P<0. 01).Administration of fluoxetine was shown to dramatically improve the depression behavior (P<0. 01) .(2) The cell proliferation [(3047. 61 ± 158. 29) vs. (1900. 33 ± 104. 10)] and survive [(2704. 21 ±154. 31) vs. (1845.33 ± 126.88)] were increased after fluoxetine administration in rats with depression (P<0. 01). (3) Fluoxetine increased mRNA expressions of Notch1 signaling components [NICD mRNA (0. 23 ±0. 01) vs. (0. 10 ±0.01), Hes1 mRNA (0. 56 ±0.04) vs. (0. 28 ±0.02), Hes5 mRNA (0. 24 ±0.02) vs. (0.10 ±0.02), Jag1 mRNA (0.82 ±0.06) vs. (0.56 ±0.03)] in the rat hippocampus compared with the CUMS group (P<0. 01 or P<0. 001). Fluoxetine enhanced protein levels of Notch1 signaling components [NICD(1.99 ±0.07) vs. (0.53 ±0.10), Hes5(0. 64 ±0. 04) vs. (0.37 ±0.09),Jag1 (2. 34 ± 0. 13) vs. (0. 68 ± 0. 17)] in the rat hippocampus compared with the CUMS group (P <0.01). Conclusion The up-regulation of the Notch1 pathway with chronic fluoxetine administration might partly contribute to increased neurogenesis in the rat hippocampus with depression.     

microRNA-183通过下调Bcl-2诱导人神经母细胞瘤细胞株SK-N-SH细胞凋亡的研究

目的研究microRNA-183对神经母细胞瘤细胞的调控作用,为神经母细胞瘤的治疗提供新策略。方法购买microRNA-183和对照microRNA,转染至人神经母细胞瘤细胞株SK-N-SH细胞,再使用MTT实验,Caspase-3活性测定试剂盒和流式检测细胞生长和凋亡的影响。合成Bcl-2siRNA,检测Bcl-2对人神经母细胞瘤细胞株SK-N-SH细胞凋亡的影响。转染Bcl-2质粒后,再转染microRNA-183至人神经母细胞瘤细胞株SK-N-SH细胞,分析Bcl-2的表达水平和人神经母细胞瘤细胞株SK-N-SH细胞的凋亡。结果转染microRNA-183降低SK-N-SH细胞的生长(P=0.005 9),发生磷脂酰丝氨酸膜表面表达(P=0.008)和Caspase-3的激活(P=0.014),Bcl-2的表达降低(P=0.015)。干扰SK-N-SH细胞中Bcl-2增强了microRNA-183诱导的细胞凋亡(P=0.005 8),而过表达Bcl-2抑制了microRNA-183诱导的细胞凋亡(P=0.007 3)。结论转染microRNA-183抑制SK-N-SH细胞的生长和增殖。microRNA-183通过下调Bcl-2而诱导SK-N-SH细胞的凋亡,提示Bcl-2可能是神经母细胞瘤潜在的候选治疗靶点。     

Interleukin-1 beta and interleukin-6 mRNA are expressed in human glioblastoma and neuroblastoma cells respectively.

We report the expression of different interleukins (IL) in four human glioblastoma and neuroblastoma cell lines. The glioblastoma cell line LI, expresses IL-1 beta and IL-6 mRNA, though not IL-2 and IL-4. The expression of the former gene is modulated by retinoic acid. Two cell clones [BE(2)-C and BE(2)-M17] as well as the neuroblastoma cell line SK-N-BE(2), from which both clones were derived, express IL-6 mRNA, but not IL-1 beta, IL-2 or IL-4. Both IL-1 beta and IL-6 cytokines are known to increase hypothalamic CRH mRNA, a gene reported to be expressed in all these cell lines. The production of both cytokines and neuropeptides indicates a complex dialogue between tumour cells and anti-tumour immunity.     

PiB对胶质瘤细胞增殖与侵袭能力的影响

目的探讨PIN1抑制剂Pi B对U87胶质瘤细胞系增殖、迁移和侵袭能力的影响。方法人胶质瘤细胞系U87常规培养,取对数生长期细胞进行试验,以1.0μmol/L Pi B处理U87细胞48 h为实验组,未经任何处理的U87细胞作为对照组。RT-PCR和Western blotting检测PIN1基因的m RNA和蛋白表达。免疫荧光检测阳性细胞数。Transwell实验检测细胞迁移和侵袭能力的改变。MTT比色法检测Pi B对细胞增殖的抑制作用。结果与对照组相比,实验组细胞PIN1基因的m RNA和蛋白表达水平明显下调,PIN1阳性细胞数明显减少,细胞抑制率明显,且与药物剂量和作用时间成相关性,细胞迁移和侵袭能力下降,差异有统计学意义（P〈0.05）。结论 Pi B可靶向抑制PIN1基因的m RNA和蛋白水平表达,进而抑制胶质瘤细胞增殖、迁移和侵袭能力。     

阻断Eag-1通道活性抑制胶质瘤细胞增殖的研究

目的 探讨阻断Eag-1通道活性对胶质瘤细胞生物活性的影响. 方法 设计3对Eag-1通道蛋白的特异性小分子干扰RNA(siRNA)并转染入U87细胞,RT-PCR及Western blotting检测其对U87细胞Eag-1 mRNA和蛋白表达的影响;将50 nmol/L siRNA1、siRNA2转染U87细胞,同时设5、10、20、30和40mmol/L奎尼丁(Eag-1通道特异性阻断剂)组和空白对照组,MTT法观察作用24、48、72 h后U87细胞增殖情况,流式细胞仪检测作用48 h后细胞周期、细胞凋亡率、胞内活性氧簇(ROS)浓度的变化. 结果 RT-PCR及Western blotting结果 显示siRNA1和siRNA2转染U87细胞12 h后细胞Eag-1 mRNA、蛋白的表达产物带明显弱于空白对照组,而siRNA3转染组产物条带虽也弱于空白对照组,但条带仍清晰;MTT检测结果 显示与空白对照组比较,50hmol/LsiRNA1、siRNA2转染组和10、20、30、40 mmol/L奎尼丁组细胞培养24、48和72 h后吸光度值均降低,差异有统计学意义(P＜0.05),奎尼丁IC50为33.7mmol/L.流式细胞仪分析显示与空白对照组比较,50nmol/L siRNA1、siRNA2转染组和33.7mmol/L奎尼丁组G1期细胞百分比、细胞凋亡率和胞内ROS水平均增加,差异有统计学意义(P＜0.05). 结论 阻断Eag-1通道活性能明显抑制胶质瘤细胞增殖,使G1期细胞比例、胞内ROS水平明显升高并诱导其凋亡.     

Instability of Notch1 and Delta1 mRNAs and reduced Notch activity in vertebrate neuroepithelial cells undergoing S-phase

Vertebrate neurogenesis is controlled through lateral inhibitory signals triggered by the Notch receptor and its ligand Delta. In the E4 chick embryo, the capacity of neural precursors to express the neurogenic genes Notch1 and Delta1 becomes reduced during S-phase, suggesting that their competence to trigger lateral inhibitory signals varies at different stages of the cell cycle. Here we show that the reduction of neurogenic gene expression during S-phase is extensive to later developmental stages and to other species; and it correlates with lower expression of lunatic Fringe and diminished capability to induce the expression of cHairy1/Hes1 and Hes5-1. We also show that the cell cycle-dependence of Notch1 and Delta1 expression is due to a remarkable decrease of mRNA stability during S-phase. These results provide evidence that the capacity of vertebrate neural precursors to express neurogenic genes and trigger lateral inhibitory signals is functionally coordinated with the cell cycle.