全反式维甲酸对大鼠C6脑胶质瘤细胞分化的影响

目的：探讨全反式维甲酸对大鼠C6脑胶质瘤细胞分化的影响。方法：建立大鼠C6脑胶质瘤模型,腹腔注射全反式维甲酸后,流式细胞仪检测细胞增殖时相变化及免疫组化检测p27Kipl蛋白和GFAP蛋白的变化。结果：流式细胞仪检测显示治疗组较对照组G1期细胞比例增加（P〈0．05）,S期细胞比例减少（P〈0．05）,免疫组化检测显示p27Kipl蛋白和GFAP蛋白的表达增加（P〈0．05）。结论：反式维甲酸可诱导大鼠C6脑胶质瘤分化,降低肿瘤恶性程度。     

全反式维甲酸对大鼠C6脑胶质瘤细胞分化的影响

目的:探讨全反式维甲酸对大鼠C6脑胶质瘤细胞分化的影响.方法:建立大鼠C6脑胶质瘤模型,腹腔注射全反式维甲酸后,流式细胞仪检测细胞增殖时相变化及免疫组化检测p27Kip1蛋白和GFAP蛋白的变化.结果:流式细胞仪检测显示治疗组较对照组G1期细胞比例增加(P<0.05),S期细胞比例减少(P<0.05),免疫组化检测显示p27Kip1蛋白和GFAP蛋白的表达增加(P<0.05).结论:反式维甲酸可诱导大鼠C6脑胶质瘤分化,降低肿瘤恶性程度.     

全反式维甲酸诱导大鼠C6脑胶质瘤细胞凋亡的实验研究

目的：探讨全反式维甲酸对大鼠C6脑胶质瘤细胞的增殖抑制及其分子机制。方法：MTT法检测全反式维甲酸作用于大鼠C6脑胶质瘤细胞后,观察其对细胞增殖抑制率的影响。流式细胞仪观察肿瘤细胞周期及凋亡率的变化。电镜观察C6细胞超微结构变化。Western blot法在不同时间点对凋亡相关基因caspase-3活性蛋白产物的表达进行了检测。结果：MTT结果表明ATRA对C6细胞的抑制作用具有时间和浓度依赖性。流式细胞仪检测证明与对照组相比,处理组C6细胞发生G1期阻滞;S、G2期细胞比例下降;细胞出现亚二倍峰,凋亡比例明显增加。电镜下全反式维甲酸作用72h后处理组C6细胞呈凋亡改变：如核固缩、染色质趋边凝聚。Western blot检测发现,处理组出现了caspase-3蛋白活性裂解片段。结论：全反式维甲酸抑制C6脑胶质瘤细胞生长,全反式维甲酸抑制脑胶质瘤的作用机理可能至少通过改变细胞周期分布、诱导凋亡来实现。     

全反式维甲酸治疗大鼠C6脑胶质瘤的实验研究

[目的]探讨全反式维甲酸治疗大鼠C6脑胶质瘤的疗效及其机制。[方法]建立大鼠C6脑胶质瘤模型,腹腔注射全反式维甲酸后,MRI检测胶质瘤的体积变化,免疫组化检测胶质纤维酸性蛋白（GFAP）的表达并观察生存期。[结果]治疗组较对照组肿瘤体积明显减小（P〈0.05）;生存期延长（P〈0.05）;GFAP表达增加（P〈0.05）。GFAP的表达同肿瘤体积呈负相关（P〈0.05）。[结论]全反式维甲酸治疗可延长荷瘤鼠生存期,其机制与上调GFAP的表达有关。     

全反式维甲酸对荷瘤Wistar大鼠生存期的影响及其机制

 目的研究全反式维甲酸对荷瘤鼠生存期的影响及其机制。方法建立wistar大鼠脑内C6胶质瘤模型，待肿瘤生长至第7天，随机将荷瘤鼠分为维甲酸治疗组和对照组。于接种后的第22天，免疫组化检测各组P27Kip1蛋白的表达并观察动物生存期。结果与对照组相比，治疗组动物p27kip1蛋白的表达增高（P〈0．05），生存期延长（P〈0．05），两者呈正相关。结论全反式维甲酸延长动物生存期的机制同上调p27kip1蛋白的表达有关。p27kip1蛋白的表达高低可作为判断胶质瘤预后的指标。     

全反式维甲酸对荷瘤Wistar大鼠生存期的影响及其机制

目的研究全反式维甲酸对荷瘤鼠生存期的影响及其机制。方法建立wistar大鼠脑内C6胶质瘤模型，待肿瘤生长至第7天，随机将荷瘤鼠分为维甲酸治疗组和对照组。于接种后的第22天，免疫组化检测各组P27Kip1蛋白的表达并观察动物生存期。结果与对照组相比，治疗组动物p27kip1蛋白的表达增高（P〈0．05），生存期延长（P〈0．05），两者呈正相关。结论全反式维甲酸延长动物生存期的机制同上调p27kip1蛋白的表达有关。p27kip1蛋白的表达高低可作为判断胶质瘤预后的指标。     

脂肪酸结合蛋白5和细胞维甲酸结合蛋白2在脑胶质瘤中的表达

目的 探讨脂肪酸结合蛋白5（fatty acid-binding protein 5,FABP5）与细胞维甲酸结合蛋白2（cellular retinoic acid-binding protein 2,CRABP2）在脑胶质瘤中的表达及与脑胶质瘤病理级别、维甲酸治疗抵抗的关系。方法 应用免疫组织化学染色法检测125例脑星型细胞瘤组织中FABP5及CRABP2蛋白表达。全反式维甲酸作用前后,以Brdu-ELISA法检测胶质瘤细胞增殖,并以实时荧光定量PCR检测胶质瘤细胞株中FABP5及CRABP2在mRNA水平的表达。结果 FABP5阳性细胞比例在WHO Ⅱ级星型细胞瘤中占（16±9）%,Ⅲ级中占（33±22）%,Ⅳ级中占（50±29）%,FABP5蛋白表达与胶质瘤病理级别呈显著正相关（P<0.05）。CRABP2阳性细胞比例在Ⅱ级星型细胞瘤中占（46±12）%,WHO Ⅲ级中占（30±15）%,Ⅳ级中占（10±9）%,CRABP2蛋白表达与胶质瘤病理级别呈显著负相关（P<0.05）。全反式维甲酸促进了胶质瘤细胞株的增殖,全反式维甲酸作用后胶质瘤细胞FABP5 mRNA表达显著上调,而CRABP2显著下调。 结论 FABP5及CRABP2的异常表达与胶质瘤恶性程度相关,并可能介导了胶质瘤细胞对维甲酸的分化抵抗效应。     

苦参碱诱导胶质瘤C6细胞凋亡及其可能的基因机制

摘 要 目的: 探讨苦参碱诱导C6胶质瘤细胞凋亡的作用特点和可能的基因作用机制。 方法：MTT法测定不同浓度苦参碱对C6细胞增殖的抑制率，求出IC50值；倒置显微镜及透射电镜观察苦参碱诱导C6细胞凋亡的形态学改变；Annexin/FITC染色流式细胞术检测不同浓度苦参碱诱导C6细胞的凋亡率；实时定量PCR芯片（Realtime PCR Array）检测苦参碱作用后C6细胞凋亡相关基因的差异表达；免疫细胞化学及Western blotting方法检测苦参碱对C6细胞caspase3表达的影响。结果：苦参碱对C6细胞增殖的抑制率随着药物剂量的增加（0.1~1.0 mg/ml）而增加（P<005），其IC50为0.715 mg/ml。倒置显微镜观察显示苦参碱可诱导胶质瘤细胞出现凋亡改变，透射电镜观察显示苦参碱诱导胶质瘤细胞出现凋亡的超微形态改变，流式细胞术检测显示胶质瘤细胞的凋亡率随着药物剂量的增加（0.2~1.0 mg/ml）而增大[(3.56±0.73)%~(27.55±1.92)%](P<0.05)。胶质瘤细胞经苦参碱作用后出现显著表达差异基因共68个，其中57个基因表达明显上调、11个基因表达明显下调，其中有22个基因与诱导凋亡的死亡受体相关，15个基因与线粒体途径有关；ICC及Western blotting检测都显示苦参碱可诱导C6胶质瘤细胞caspase3表达的上调(P<0.05)。 结论：苦参碱能诱导C6胶质瘤细胞的凋亡，其机制与死亡受体途径和线粒体途径的众多基因有关。     

急性早幼粒细胞白血病细胞黏附分子表达与维甲酸综合征的关系

目的：观察急性早幼粒细胞白血病细胞在诱导分化中黏附分子的表达与维甲酸综合征的关系。方法：流式细胞仪方法进行细胞表面标记检测，标本来自于16例急性早幼粒细胞白血病患者的骨髓标本．标本采集时间为应用全反式维甲酸治疗前、用药后7d、14d及28d。结果：16例患者在诱导缓解过程中有3例患者出现维甲酸综合征，与CD11b表达时间的相关性为7d：r=0．884，P=-0．000；14d：r=0．541，P=0．030；28d：r=0．38.P=0．280。结论：急性早幼粒白血病细胞在全反式维甲酸诱导缓解过程中早期、同步表达细胞黏附分子CD11b与维甲酸综合征的发生密切相关。     

冷冻诱导大鼠C6脑胶质瘤细胞发生凋亡的实验研究

目的：通过观察大鼠C6脑胶质瘤经冷冻后出现细胞凋亡，探讨冷冻对肿瘤的杀伤机制。方法：建立大鼠C6脑胶质瘤颅内接种模型，通过TUNEL法及流式细胞仪分别检测其冷冻后凋亡数目及凋亡率。结果：肿瘤经冷冻后，其中心出现坏死，而周边细胞呈现典型的凋亡形态学改变。TUNEL法检测冻后24、48和72h凋亡数目0．323、0．00167和0．037；流式细胞仪检测其冻后24、48和72h凋亡率与对照组相比，P值分别为0．0254、0．0064和0．031；冻后各时间点凋亡数目及凋亡率均显著高于对照组，P〈0．05。其中冻后48h改变尤其明显，P〈0．01，峰值时间出现在冷冻24～48h。结论：诱导细胞凋亡也是冷冻杀伤肿瘤细胞的重要机制之一，但其发生的分子机制有待于进一步深入研究。     

蒿甲醚抗SD大鼠原位脑胶质瘤血管生成的 实验

 目的探讨蒿甲醚（Artemether）抗SD大鼠原位脑胶质瘤血管生成作用。方法采用四甲基偶氮唑蓝(MTT)法测定不同浓度蒿甲醚对大鼠C6脑胶质瘤细胞株的生长抑制作用,计算半数抑制浓度(IC₅₀)。采用立体定位仪在SD大鼠大脑皮质层接种C6脑胶质瘤细胞（1×10⁶/μl）40只,雌、雄各半;随机分为5组,每组8只。在接种第3天后,各组采用灌胃给药法连续给药10天。于接种后的第20天解剖大鼠,经活体左心室灌注4%多聚甲醛,固定肿瘤的全脑标本。在大鼠脑部接种穿刺点做冠状切口,按垂直和水平方向测量肿瘤大小。肿瘤体积=a²bπ∕6(a为肿瘤的短径,b为肿瘤的长径)。全脑标本用4%多聚甲醛固定,肿瘤组织做病理观察,免疫组化方法检测移植瘤组织微血管密度。结果各实验组血管计数分别为Ⅰ组（39±4）,Ⅱ组（29±6）,Ⅲ组（12±8）,Ⅳ组（10±5）,生理盐水组为（52±7）。各实验组血管计数均明显少于生理盐水对照组,差异有统计学意义(分别P<0.05, P<0.01)。各实验组SD大鼠原位脑胶质瘤体积较对照组显著减小。结论在一定剂量范围内,蒿甲醚具有明显抑制SD大鼠原位脑胶质瘤血管生成作用;蒿甲醚抑制原位脑胶质瘤生长和转移的机制之一是透过血脑屏障抑制脑胶质瘤血管生成。     

冷冻治疗抑制大鼠C6脑胶质瘤增殖的实验研究

目的：研究冷冻治疗对大鼠C6脑胶质瘤的增殖抑制作用及其机制。方法：建立大鼠C6脑胶质瘤模型，冷冻治疗后免疫组化检测CyclinD1蛋白的表达，流式细胞仪检测细胞周期的变化，磁共振检测肿瘤体积变化。结果：与对照组比较，冷冻治疗后CyclinD1蛋白的表达下调，细胞周期出现G1期阻滞，肿瘤体积缩小。结论：冷冻治疗可抑制大鼠C6脑胶质瘤的增殖，其机制与下调CyclinD1蛋白的表达后，肿瘤细胞周期发生G1期阻滞有关。     

Induction of differentiation and tetraploidy by long-term treatment of C6 rat glioma cells with erucylphosphocholine

Induction of differentiation represents a promising concept for chemotherapy of malignant gliomas, which are often refractory even to the combined treatment with surgery, irradiation and chemotherapy. Since anti-neoplastic alkylphosphocholines can induce differentiation of leukemic cell lines, the effects of the intravenously applicable alkylphosphocholine-derivative erucylphosphocholine (ErPC) on proliferation, morphology and differentiation of the rat glioma cell line C6 was examined in vitro. Short-term exposure to ErPC induced accumulation of the cells in the G2/M-phase of the cell cycle and apoptotic cell death. In contrast, continuous exposure of C6 rat glioma cells to sublethal ErPC doses (30 and 50 microM) caused both the formation of a slower growing tetraploid cell population and astrocytic differentiation. No resistance to in vivo obtainable ErPC concentrations was observed during this treatment. We conclude that ErPC-induced differentiation might be beneficial for a long-term adjuvant chemotherapy of low grade glioma.     

GM-CSF在替莫唑胺抗高级别胶质瘤中的作用

目的 探讨粒-巨噬细胞集落刺激因子（granulocyte-macrophage colony-stimulating factor, GMCSF）在替莫唑胺（temozolomide, TMZ）抗高级别胶质瘤中的作用及机制。方法 选取6例高级别胶质瘤患者来源的肿瘤组织培养肿瘤细胞,待细胞状态稳定后采用MTT法进行细胞增殖毒性实验,流式细胞仪检测细胞周期变化和细胞凋亡情况,甲基化特异性PCR和免疫组织化学染色法分别检测六氧甲基鸟嘌呤DNA甲基转移酶（O6-methylguanine-DNA methyltransferase, MGMT）基因启动子甲基化状态和MGMT蛋白表达水平。 结果 MTT实验显示,GM-CSF处理组的细胞存活率与对照组相比均有不同程度的增加。MGMT基因启动子甲基化的3例细胞,GM-CSF+TMZ组的细胞存活率比TMZ组显著降低（P<0.05）,另3例MGMT启动子非甲基化细胞,GM-CSF+TMZ组与TMZ组相比,其存活率差异均无统计学意义（P>0.05）。6例细胞GM-CSF组的G1期细胞比例均比对照组降低,而S期细胞比例GMCSF处理组较对照组显著增加（P<0.05）。流式细胞仪凋亡检测显示,MGMT启动子甲基化的3例细胞,GM-CSF+TMZ组凋亡率与单药TMZ组凋亡率相比均显著增加（P<0.05）,而MGMT非甲基化细胞GM-CSF+TMZ组与单药TMZ组凋亡率相比无统计学差异。结论 GM-CSF可通过诱导高级别胶质瘤细胞快速进入细胞周期,显著提高TMZ对MGMT基因启动子甲基化的高级别胶质瘤细胞的杀伤作用,而对MGMT基因启动子非甲基化高级别胶质瘤细胞的作用不明显。     

全反式维甲酸与单纯疱疹病毒胸苷激酶基因对胶质瘤的协同杀伤作用

背景与目的：细胞间缝隙连接通讯（gap junctional intercellular communication,GJIC)是介导单纯疱疹病毒胸苷激酶（herpes simplx virus thymidine kinase,HSV-tk)基因治疗中旁观者效应的重要机制。全反式维甲酸(all-trans-retinotic acid,ATRA)或能通过上调胶质细胞GJIC和抑制肿瘤细胞生长的双重作用,促进HSV-tk基因治疗的疗效。本研究的目的在于证实HSV-tk和ATRA联合应用对胶质瘤细胞的协同杀伤作用。方法：分别以1μmol/L、10μmol/L、100μmol/L3种浓度的ARTA作用于培养的大鼠C6胶质瘤细胞,并研究ATRA对C6胶质瘤细胞分化、增殖、GJIC及connexin43(Cx43)基因转录等方面的影响。将C6细胞与稳定表达HSV-tk基因的C6tk细胞按不同比例混合,分别在含GCV和不同浓度ATRA或仅含GCV的培养基中培养,并于药物作用7天后用MTT法检测旁观者效应。结果：经3种浓度的ARTA作用后,C6胶质瘤细胞均显示出细胞分化的形态学特征。C6细胞的增殖也明显受到ATRA的抑制,绝大多数的活细胞都静止于G1期,100μmol/L ATRA对C6细胞增殖的抑制尤其明显,并诱导细胞凋亡。经3种浓度的ATRA分别作用后,C6细胞的GJIC也明显提高,而Cx43基因的转录未受明显影响。旁观者效应实验的结果显示,3种浓度的ATRA均可明显增强旁观者效应。结论：联合应用ATRA与HSV-TK基因治疗可以产生显著的杀伤胶质瘤细胞的作用。     

Evidence for cell cycle phase-specific initiation of a program of HL-60 cell myeloid differentiation mediated by inducer uptake

The question of whether the initial regulatory event, which directs an uncommitted precursor cell toward terminal differentiation, is cell cycle phase specific was examined using the human promyelocytic leukemia cell line, HL-60. While the HL-60 system does not reflect all of the features of normal hematopoiesis, it does provide a relatively well-defined in vitro experimental system which can be useful for examining aspects of the differentiation process. HL-60 cells were induced to undergo myeloid differentiation by retinoic acid. The subsequent differentiation kinetics of HL-60 populations initially enriched in different cell cycle phases was measured. This was compared to the cellular uptake of retinoic acid as a function of cell cycle position. If the initial differentiation-regulating event were cell cycle phase independent, then the kinetics of differentiation would be independent of the cell cycle status of the initial population. Flow cytometric cell sorting, based on cellular narrow angle and orthogonal light scatter intensity spectra, was used to select G1-enriched and S + G2 + M-enriched cell populations without pharmacological perturbation. These two populations were each induced to undergo myeloid differentiation with 10(-6) M beta-all-trans-retinoic acid. The kinetics of G1/0 arrest associated with terminal cell differentiation, as well as phenotypic differentiation, assayed by development of oxidative metabolism, was measured for both populations. The kinetics of differentiation differed for the two populations, indicating that the initial differentiation-regulating event was cell cycle phase specific. For both of the initial cell populations, significant phenotypic differentiation followed approximately 24 hr after enrichment in the relative number of S-phase cells. When exponentially proliferating HL-60 cells were exposed to a 1-hr pulse of 10(-5) M [3H]retinoic acid and then flow cytometrically sorted by DNA content, cells in late S + G2 + M had an approximately 10-fold higher uptake than cells in G1 or early S. The results indicate that cellular regulation of myeloid differentiation first becomes responsive to the inducer, retinoic acid, in S phase when uptake is enhanced.     

The tyrosine kinase inhibitor ZD6474 inhibits tumour growth in an intracerebral rat glioma model

Malignant glioma is characterised by extensive neovascularisation, principally influenced by vascular endothelial growth factor (VEGF). ZD6474 is a potent inhibitor of VEGF-R2 tyrosine kinase activity, but with additional inhibitory effects on other growth factors. In this study, we have investigated the effects of ZD6474 with regard to tumour growth, neovascularisation, proliferation and apoptosis in the intracerebral rat glioma model, BT4C. ZD6474 (50 and 100 mg kg(-1)) was given as a daily oral gavage. Animals were killed on day 19 and tumour volume was measured. Sections were stained for factor VIII, Ki-67 and for apoptosis. The ability of ZD6474 to inhibit cell growth directly was examined in vitro, using the glioma cell line BT4C and the transformed rat brain endothelial cell line RBE4. Cell growth was analysed with fluorometric microculture cytotoxicity assay to quantify the cytotoxic effects. ZD6474 significantly decreased tumour volume compared to controls. Microvascular density increased after treatment with ZD6474, and tumour cell proliferation index was reduced. There was also an increase in tumour cell apoptosis. In vitro, the growth of both cell lines was significantly reduced. The results reported justify further experimental investigations concerning the effects of ZD6474 in malignant glioma alone or in combination with other modalities.     

Combination celecoxib and temozolomide in C6 rat glioma orthotopic model

The purpose of this study was to determine whether a combination treatment of temozolomide with celecoxib is effective in the rat orthotopic glioma model. After stereotactic injection of C6/LacZ rat glioma cells into the Sprague Dawley rat brain, the rats were randomly assigned to four treatment groups [group 1, control treatment; group 2, celecoxib (25 mg/kg p.o. everyday) alone; group 3, temozolomide (7.5 mg/kg i.p. for 5 days at 2nd week) alone; group 4, a combination of celecoxib and temozolomide]. Rats were sacrificed 18 days after treatment, and the body weight, tumor volume, tumor cell proliferation, microvessel densities, and apoptosis were evaluated. There was a significant reduction of tumor volume in combination group compared to control or single-agent therapy. The median tumor volume was estimated to be 111.5 mm(3) (control), 65.0 mm(3) (celecoxib), 71.8 mm(3) (temozolomide) and 18.7 mm(3) (combination). In the combination group, there was increased tumor cell apoptosis as well as decreased microvessel density and tumor cell proliferation relative to the control and single-agent therapy (P<0.05). Collectively, the data suggest that the combination celecoxib and temozolomide may provide a novel and effective approach to the treatment of glioblastoma.     

外源性RGS16基因稳定转染对胶质瘤C6细胞生长的影响

背景与目的:有研究表明野生型p53可以诱导RGS16表达,而RGS16可能与胶质瘤的发生有关。本研究旨在探讨RGS16基因转染对大鼠胶质瘤C6细胞生长的影响。方法:构建真核表达载体pIRES2-EGFP-RGS16,脂质体法转染C6细胞,经G418筛选后荧光显微镜观察细胞中增强型绿色荧光蛋白(enhancedgreenfluorscentprotein,EGFP)的表达,RT-PCR证实RGS16的mRNA表达,免疫细胞化学方法检测细胞中RGS16蛋白表达。最后利用流式细胞仪、生长曲线、平板克隆形成等方法研究RGS16基因对胶质瘤细胞周期、生长及增殖的影响。结果:成功构建了稳定表达RGS16和表达空载体的细胞系C6-RGS16、C6-GFP。C6-RGS16、C6-GFP和C6经流式细胞仪检测S期的细胞比例分别为28.5%、18.9%和14.3%(P<0.05);生长曲线表明C6-RGS16生长的速度明显快于C6-GFP及C6,但其平板克隆形成率分别为12%、25%和25%(P<0.05)。结论:RGS16促进C6细胞周期从G1期向S期过渡,加快细胞生长速度,但是并不促进细胞克隆形成。