表达VASH1基因的人脑胶质瘤U-87MG细胞对化疗药物敏感性的变化

目的 探讨表达人血管生成抑制因子１（VASH1）的人脑胶质瘤U-87MG细胞对化疗药物的敏感性变化。方法 构建针对VASH1的慢病毒载体pGCL-GFP-VASH1,经测序鉴定后转染293T细胞,筛选出适合浓度的慢病毒转染人脑胶质瘤U-87MG细胞,荧光显微镜下检测转染效率;通过RT-PCR和Western blot分析U-87MG细胞VASH1 mRNA和蛋白表达水平;用CCK-8法检测U-87MG细胞在化疗药物顺铂和替莫唑胺作用下的存活率。流式细胞仪检测U-87MG细胞凋亡。结果 成功构建pGCL-GFP-VASH1慢病毒载体,并成功转染U-87MG细胞,转染率达70％以上;RT-PCR和Western blot结果证实转染VASH1慢病毒载体的U-87MG细胞表达VASH1 mRNA和蛋白。在顺铂或替莫唑胺作用下,表达VASH1的U-87MG细胞存活率均较未表达VASH1的U-87MG细胞明显降低（P<0.01）,而且U-87MG细胞凋亡率明显增加（P<0.01）。结论 VASH1慢病毒载体转染U-87MG细胞可使其稳定表达VASH1,并提高人脑胶质瘤U-87MG细胞对化疗药物敏感性、增加细胞凋亡率。     

Insulin-like growth factor-I decreased etoposide-induced apoptosis in glioma cells by increasing bcl-2 expression and decreasing CPP32 activity

AIMS: In a variety of tumors, the susceptibility of the tumor cells to apoptotic cell death following chemotherapy is a major determinant of therapeutic outcome. Gliomas are resistant to most chemotherapeutic agents, and its mechanism is not known in detail. In an attempt to understand the mechanism of chemo-resistance, we investigated the roles of insulin-like growth factor-I (IGF-I), IGF-I receptors (IGF-IR), and their relationship with the apoptotic response of two glioma cell lines to etoposide, a chemotherapeutic agent for malignant gliomas. METHODS: Two human glioma cell lines, U-87MG and KNS-42, were used. Etoposide-induced cell growth inhibition was quantified using a modified MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide), colorimetric assay. Hoechst 33258 staining, DNA fragmentation assay, and western blot were used for the evaluation of apoptosis. ApoAlert caspase assay was used for measuring the activity of caspase-3 (CPP32) and interleukin-1 beta -converting enzyme (ICE) protease. In addition, the effect of IGF-IR antisense was tested in U-87MG and KNS-42 glioma cell lines. RESULTS: Etoposide inhibited the growth of U-87MG and KNS-42 cells in a concentration-dependent manner. Etoposide increased the expression of wild-type p53, activated CPP32 (but not ICE) activity, and induced apoptosis in these cells. IGF-I prevented etoposide-induced apoptosis by increasing the expression of bcl-2 and decreasing the activity of CPP32. IGF-IR antisense enhanced the apoptotic effect of etoposide. CONCLUSIONS: IGF-I decreased etoposide-induced apoptosis in glioma cells by increasing the expression of bcl-2 and decreasing the activity of CPP32. The antisense of IGF-IR increased etoposide-induced apoptosis. The anti-apoptotic effect of IGF-I and IGF-IR might be related to the chemo-resistance of glioma to chemotherapeutic agents.     

Monoclonal antibody against human glioblastoma multiforme (U-87MG) immunoprecipitates a protein of molecular mass 38 kDa and inhibits tumor growth in nude mice

A monoclonal antibody 6DS₁ against a human glioblastoma multiforme cell line U-87MG recognizes a tumor-specific, cell surface antigen of human glioblastoma cell lines. Partial cross-reactivity is observed with two human neuroblastoma cell lines, SK-N-SH and SK-N-MC, with little or no reactivity towards a rat glioma cell line C6 or normal human adult and fetal brain tissues. The antibody recognizes an antigen of molecular mass 38 kDa as inferred from Western blot analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the immunoprecipitate. The monoclonal antibody 6DS₁ inhibits both the attachment to substratum and growth of U-87MG cells. It strongly cross-reacts with xenotransplants of U-87MG cells and inhibits tumorigenesis (subcutaneous implants of U-87MG cells) in nude mice.     

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。     

Differentiation inducing effects of vesnarinone on human glioma cells

The effects of 3,4-dihydro-6-[4-(3,4-dimethoxybenzoyl)-1-piperazinyl]-2(1H)-quinolinone (vesnarinone) on the growth of glioma cells were examined in vitro. Vesnarinone at a dose of 100 mug/ml suppressed the growth of four different glioma cell lines, U-251MG, U-373MG, U-87MG and A-172, by approximately 50%, with an elongation of the cytoplasmic process on day 5 of culture. The long-term culture of U-87MG with 10 mug/ml of vesnarinone was continued up to day 34. Although growth suppression was approximately 25% on day 5, it reached over 95% on day 34. An increase in the cyclic adenosine monophosphate content of glioma cells cultured with vesnarinone was observed by enzyme-linked immunosorbent assay (ELISA). The accumulation of glial fibrillary acidic protein was observed to occur with vesnarinone by ELISA. These findings suggest that vesnarinone suppressed the growth and induced differentiation of glioma cells in vitro.     

慢病毒介导的RNAi下调miR-221抑制U87胶质瘤细胞生长的研究

目的 探讨下调miR-221对U87胶质瘤细胞生长的影响及可能的作用机制.方法 构建靶向miR-221的siRNA慢病毒表达载体并感染U87胶质瘤细胞,应用原位杂交、定量PCR和Western blot法检测干扰后细胞内miR-221及p27蛋白表达变化,MTT法检测细胞增殖,流式细胞仪检测细胞凋亡及周期变化,Transwell法检测细胞侵袭力改变.结果 成功构建了靶向miR221的siRNA慢病毒表达载体,该载体能有效抑制内源性miR-221的表达,同时上调p27蛋白的表达,同时抑制细胞生长,诱导凋亡,降低细胞的侵袭力.结论 成功构建靶向miR-221的RNAi慢病毒表达载体,该载体能够有效抑制miR-221的表达并抑制U87胶质瘤细胞的生长,为以miR-221为靶点的胶质瘤基因治疗的后续研究奠定基础.     

Sphingosine kinase-1 expression correlates with poor survival of patients with glioblastoma multiforme: roles of sphingosine kinase isoforms in growth of glioblastoma cell lines

Sphingosine-1-phosphate is a bioactive lipid that is mitogenic for human glioma cell lines by signaling through its G protein-coupled receptors. We investigated the role of sphingosine-1-phosphate receptors and the enzymes that form sphingosine-1-phosphate, sphingosine kinase (SphK)-1, and -2 in human astrocytomas. Astrocytomas of various histologic grades expressed three types of sphingosine-1-phosphate receptors, S1P1, S1P2, and S1P3; however, no significant correlation with histologic grade or patient survival was detected. Expression of SphK1, but not SphK2, in human astrocytoma grade 4 (glioblastoma multiforme) tissue correlated with short patient survival. Patients whose tumors had low SphK1 expression survived a median 357 days, whereas those with high levels of SphK1 survived a median 102 days. Decreasing SphK1 expression using RNA interference or pharmacologic inhibition of SphK significantly decreased the rate of proliferation of U-1242 MG and U-87 MG glioblastoma cell lines. Surprisingly, RNA interference to knockdown SphK2 expression inhibited glioblastoma cell proliferation more potently than did SphK1 knockdown. SphK knockdown also prevented cells from exiting G1 phase of the cell cycle and marginally increased apoptosis. Thus, SphK isoforms may be major contributors to growth of glioblastoma cells in vitro and to aggressive behavior of glioblastoma multiforme.     

Differential control of VEGF synthesis and secretion in human glioma cells by IL-1 and EGF.

VEGF (vascular endothelial growth factor), one of the most potent angiogenic factors, has recently been identified as an inducer of neoangiogenesis in many tumors including gliomas. VEGF itself appears to be regulated through different pathways. Since malignant gliomas frequently show EGF receptor amplification and express IL-1, a pivotal regulatory cytokine involved in angiogenesis, we analyzed interactions between EGF/EGF receptor and IL-1/IL-1 receptor and VEGF in the established glioblastoma cell lines U-87 MG and A-172. Basal VEGF expression was an order of magnitude higher in U-87 MG compared to A-172. IL-1 caused a fast and strong increase of VEGF secretion in U-87 MG which appeared to harbor an intracellular VEGF pool for enhanced exocytosis. The IL-1 receptor antagonist (IL-1-ra) reversed this effect suggesting an IL-1 receptor-associated mechanism. In contrast, VEGF secretion could not be increased by exogenous IL-1 exposure in A-172, which apparently lacked an intracellular VEGF pool for augmented exocytosis. However, IL-1-ra treatment alone caused a significant reduction of basal VEGF secretion in both U-87 MG and A-172. This suggests that baseline secretion of VEGF involves IL-1 receptor activation by endogenously produced IL-1. EGF also stimulated the secretion of VEGF into the cell supernatant. However, this effect, observed in both U-87 MG and A-172, was delayed and only occurred following replenishment of the intracellular VEGF pool. EGF upregulated the amount of VEGF mRNA. In general, the effects of IL-1 and EGF on VEGF were additive, suggesting independent mechanisms. Since IL-1 appears to be involved in VEGF secretion in glial tumors through an autocrine/paracrine mechanism, recombinant human IL-1-ra may evolve as a new agent for anti-angiogenic glioma therapy.     

替莫唑胺通过TFRC抑制U87胶质瘤细胞增殖和侵袭

目的 探讨转铁蛋白受体（TFRC）在替莫唑胺抑制U87胶质瘤细胞增殖和侵袭中的作用。方法 体外培养U87胶质瘤细胞,加入50 μmol/L、100 μmol/L和200 μmol/L替莫唑胺作用;构建control siRNA、siTFRC转染U87细胞沉默TFRC表达,构建pcDNA3.1空载体、pcDNA3.1/TFRC转染U87细胞过表达TFRC;利用CCK-8方法检测U87细胞增殖;利用Transwell小室实验检测U87细胞侵袭能力;实时荧光定量PCR和免疫印迹法检查U87细胞TFRC mRNA和蛋白表达。结果 与空白组相比,替莫唑胺处理后,U87细胞的增殖和侵袭能力显著下降（P<0.05）,TFRC mRNA和蛋白表达水平显著降低（P<0.05）,当替莫唑胺浓度为200 μmol/L时,对U87细胞的抑制能力最强（P<0.05）。当利用pcDNA3.1/TFRC过表达U87细胞TFRC处理后,替莫唑胺对U87细胞增殖和侵袭的抑制能力显著下降（P<0.05）;而当利用siTFRC沉默U87细胞TFRC表达后,替莫唑胺对U87细胞增殖和侵袭的抑制能力显著增强（P<0.05）。结论 替莫唑胺可能通过抑制TFRC的表达而抑制U87胶质瘤细胞的增殖和侵袭。     

Gangliosides have a bimodal effect on DNA synthesis in U-1242 MG human glioma cells

GM1, GD1a, and GT1b inhibit both PDGF-stimulated and serum-stimulated DNA synthesis in Swiss 3T3 cells and the human glioma cell line U-1242 MG in a dose-dependent manner. The ganglioside inhibitory effect is counteracted in a dose-responsive fashion by serum such that ganglioside-induced inhibition is essentially abolished in 10% serum. Because of the potentially important role that gangliosides play in growth regulation of human gliomas, this phenomenon was studied in detail using U-1242 MG cells. Stimulation of DNA synthesis by low doses of serum in U-1242 MG cells is inhibited in a dose-responsive fashion by ganglioside GM1. However, serum itself counteracts the inhibitory effect of ganglioside in a dose responsive way. Kinetic analyses demonstrate that GM-1 competes with some components of serum for sites on U-1242 MG cells (Kb of GM1 =12.5 μM). On the other hand, GM1, GD1a, and GT1b stimulate DNA synthesis in quiescent U-1242 MG cells in both sparse and confluent conditions, indicating that ganglioside-stimulated DNA synthesis is dependent on the phase of cellular growth rather than cellular density. This growth stimulatory effect of gangliosides is more potent on quiescent, confluent cells than quiescent, sparse cells. These results demonstrate that exogenously added gangliosides can have opposite (bimodal) effects on progression of human glioma cells through the cell cycle depending upon the growth phase of the cells. © 1995 Wiley-Liss, Inc.     

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.     

Effects of inducible glial fibrillary acidic protein on glioma cell motility and proliferation

We have studied the effect of induced glial fibrillary acidic protein (GFAP) on motility, cell morphology, and proliferation of two originally GFAP-negative human glioma cell lines. Glioma cell lines U-1242 MG and U-251 MG sp subclone 3A were transfected with a vector system that allows for an inducible GFAP expression. This experimental system creates an "on/off" situation in which GFAP expression is suppressed by tetracycline. Inducible expression of GFAP in the absence of tetracycline was confirmed by immunofluorescence staining and Northern and Western blotting. The study showed that forced GFAP expression resulted in an inhibition of cell motility measured as the phagokinetic track area of individual cells seeded sparsely on a surface covered with gold particles. It also resulted in a change in cell morphology, with extended cell processes, and it was associated with a low fraction of cells in S-phase. We conclude that the down-regulation of GFAP expression that is often seen in gliomas in vivo may be an important parameter of tumor progression related mainly to the motile and thereby invasive properties of malignant glioma cells.     

Physiology of transformed glial cells

Much of our present knowledge of glial cell function stems from studies of glioma cell lines, both rodent (C6, C6 polyploid, and TR33B) and human (1321N1, 138MG, D384, R-111, T67, Tp-301MG, Tp-483MG, Tp-378MG, U-118MG, U-251MG, U-373MG, U-787MG, U-1242MG, and UC-11MG). New methods such as patch clamp and Ca²⁺ imaging have lead to rapid progress the last few years in our knowledge about glial cells, where an unexpected presence and diversity of receptors and ion channels have emerged. Basic mechanisms related to membrane potential and K⁺ transport and the presence of voltage gated ion channels (Na⁺, inwardly rectifying K⁺, Ca²⁺ activated K⁺, Ca²⁺, and Cl^? channels) have been identified. Receptor function and intracellular signaling for glutamate, acetylcholine, histamine, serotonin, cathecolamines, and a large number of neuropeptides (bradykinin, cholecystokinin, endothelin, opioids, and tachykinins) have been characterized. Such studies are facilitated in cell lines which offer a more homogenous material than primary cultures. Although the expression of ion channels and receptors vary considerably between different cell lines and comparative studies are rare, a few differences (compared to astrocytes in primary culture) have been identified which may turn out to be characteristic for glioma cells. Future identification of specific markers for receptors on glial and glioma cells related to cell type and growth properties may have great potential in clinical diagnosis and therapy. © 1995 Wiley-Liss, Inc.     

HSP70调节ATO诱导胶质瘤细胞死亡的实验研究

目的通过调节热休克蛋白70(HSP70)的表达,观察HSP70在三氧化二砷(ATO)诱导胶质瘤细胞U251MG死亡中的作用。方法台盼蓝染色用来评价细胞的活性,免疫印迹分析HSP70和Caspase-3表达情况,进一步使用热休克蛋白抑制剂KNK437和热激的方法调节HSP70的水平,观察ATO在不同HSP70水平对U251MG死亡的影响。结果 ATO诱导U251MG细胞的死亡,伴有HSP70的表达;应用KNK437明显增加ATO诱导U251MG细胞的死亡和凋亡蛋白Caspase-3表达;热激明显抑制ATO诱导U251MG细胞的死亡。结论 HSP70在ATO诱导胶质瘤细胞U251MG死亡中起保护作用,KNK437可能在ATO治疗胶质瘤中起协同作用。     

短发夹RNA靶向抑制suvivin基因在人胶质瘤细胞U251中的表达

目的构建表达靶向抑制survivin基因的三个短发夹结构(shRNA)的RNA干扰载体,使其在胶质瘤细胞发挥干扰效应。方法在survivin全长序列中选取设计3条19个核苷酸靶序列,2条反向重复序列,间以9个核苷酸的茎环序列,加上对应酶切位点,形成3条shRNA的DNA模板,分别克隆到3个干扰载体pG1,pG2和pG3上：采用分布酶切连接的方法,分别将U6启动子以及下游的后2个shRNA模板酶切下来,依次连接到pG1对应酶切位点,构建出含有3条shRNA模板且能独立编码shRNA的重组干扰载体pGenesil-survivin,测序鉴定：将干扰质粒导入到胶质瘤细胞U251,分别采用RT-PCR以及Western Bloting从mRNA和蛋白水平检测干扰后效果。结果重组的干扰载体含有3条正确的shRNA模板；RT-PCR以及Western Blotting检测显示,survivin的mRNA转录水平以及蛋白水平的表达均得到显著抑制。结论编码3条shRNA的干扰载体pGenesil-survivin介导的RNA干扰技术(RNAi)可以显著的靶向抑制survivin基因在人胶质瘤细胞U251中的表达。     

吉非替尼对表达PTEN的胶质瘤细胞株U87细胞增殖活性的影响

目的探讨表皮生长因子受体抑制剂吉非替尼对表达磷酸酶一张力蛋白同源物（FrEN）的胶质瘤细胞株U87细胞增殖活性的影响。方法将人脑恶性胶质瘤细胞株U87细胞（PTEN缺失）置人DMEM中培养,根据转染质粒不同分为空白组（不转染任何质粒）、空载体组（转染pCDNA3．1载体）和PTEN组（转染pCDNA3．1-PTEN质粒）,采用5-溴脱氧尿嘧啶核苷（Brdu）,碘化丙碇双掺入法分析细胞DNA合成水平,采用流式细胞仪分析细胞周期,采用蛋白免疫印迹法分析蛋白表达。结果与空白组和空载体组相比,PTEN组PTEN蛋白表达水平明显升高,而磷酸化Akt蛋白表达水平明显降低。PTEN组BrdU阳性细胞比例[（13．5±2．7）％]明显低于空白组[（39．5±4．2）％;P〈0．05]和空载体组为[（40．7±5．1）％;P〈0．05]。PTEN组GJG。期细胞比例[（80．2±6．6）％]明显高于空白组[（43．2±5．2）％;P〈0．05]和空载体组[（41．1＋4．7）％;P〈0．05],而s和G2,M期细胞比例[分别为（35．7±3．7）％和（21．1±2．1）％]明显低于空白组[分别为（36．5±4．1）％和（22．4±1．9）％;P〈0．05]和空载体组[分别为（12．7＋2．O）％和（7．1±1．1）％;P〈0．051。结论高表达PTEN蛋白能够增强胶质瘤细胞株U87细胞对吉非替尼的敏感性。