顺铂缓释剂瘤内治疗大鼠C6胶质瘤实验观察

观察顺铂缓释剂瘤内用药治疗大鼠Ｃ６胶质瘤的生物学效应。方法用几丁质做载体,与顺铂制成缓释剂型,进行瘤内植入治疗大鼠脑胶质瘤。结果瘤内给予１ｍｇ／ｍ＾２顺铂缓释剂无毒性,瘤内铂浓度比全身用药高２－５０倍,维持时间达１月以上,血铂浓度比全身用药低１０－６０倍,荷瘤鼠生存时间较单纯顺铂液瘤内或全身用药明显延长。     

Celecoxib诱导C6胶质瘤细胞凋亡的研究

目的探讨Celecoxib(塞来昔布)诱导C6胶质瘤细胞凋亡的作用。方法应用吖啶橙、透射电镜和流式细胞仪(FCM)检测方法。结果通过吖啶橙及电镜检查发现Celecoxib 50μmol／L组细胞凋亡明显多于12．5μmol／L组，FCM检测Celecoxib 50μmol／L组细胞凋亡为15．32％，12．5μmol／L组凋亡为5．83％，不同浓度的Celecoxib对C6胶质瘤细胞有增殖抑制及诱导凋亡的作用，凋亡随药物浓度增加而升高。结论Celecoxib对C6胶质瘤细胞呈剂量依赖性抑制及诱导凋亡的作用．对胶质瘤治疗有重要意义。     

三氧化二砷诱导C6胶质瘤细胞凋亡实验研究

目的：研究三氧化二砷（As2O3）在体外是否具有抗鼠胶质瘤作用,并对其作用机制进行初步探讨。方法：应用不同浓度As2O3,且在不同时间点分别处理C6胶质瘤细胞株及原代培养的正常鼠神经胶质细胞,采用甲基噻唑基四唑（MTT）法,观察As2O3对C6胶质瘤细胞株及正常鼠神经胶质细胞生长的影响,以透射电镜、Hoechst33342和碘化丙啶（PI）双重荧光染色检测两种细胞凋亡的形态变化,并用异硫氰酸荧光素（FITC）标记的Annexin-V和PI双标记法通过流式细胞仪检测细胞凋亡率。结果：MTT法发现,As2O30.5～8.0μmol/L的浓度均可显著抑制C6胶质瘤细胞株的生长,而对正常原代神经胶质细胞株的抑制作用较弱。经As2O3作用后,透射电镜、Hoechst33342/PI双染荧光均观察到C6胶质瘤细胞发生了显著的凋亡形态改变;运用Annexin-V-FITC/PI双标记法在流式细胞仪检测显示,随As2O3浓度的增大和时间的延长,C6胶质瘤细胞株的凋亡率明显上升,而正常神经胶质细胞的凋亡率要明显小于C6胶质瘤细胞株。结论：As2O3在体外可显著抑制C6胶质瘤细胞株生长,其机制与诱导肿瘤细胞凋亡有关,且凋亡率随As2O3作用的时间和剂量的增加而增加。但As2O3对正常神经胶质细胞生长的抑制作用较弱,提示As2O3抑制细胞生长的作用具有一定的选择性。     

光动力联合顺铂治疗大鼠脑胶质瘤的生存期观察

目的 探讨光动力（PDT）联合顺铂治疗大鼠脑恶性胶质瘤的杀伤效应、机制及对大鼠生存期影响.方法 健康Wistar雄性大鼠30只,体重280～300g,建立C6胶质瘤模型,2周后随机分为3组,对照组、顺铂治疗组、PDT联合顺铂治疗组,分别进行生存观察和生存分析.结果 大鼠平均生存时间：对照组（38.2±3.5）d,顺铂组（39.6±4.0）d,PDT联合顺铂组（41.9±4.0）d.联合顺铂组与对照组及顺铂组比较,生存分析曲线差异有显著性（P〈0.05）.结论 光动力（PDT）联合顺铂治疗对大鼠脑胶质瘤有明显的杀伤和抑制作用,光动力开放血脑屏障、血瘤屏障后为化疗药物有效作用于肿瘤组织提供新途径,延长大鼠生存期.     

反义IGF-IR基因片段诱导C6胶质瘤细胞凋亡

一、材料与方法1 .寡核苷酸的设计、合成 :根据ＩＧＦ ＩＲｃＤＮＡ序列 ,设计的正义寡核苷酸为 5 Ａ Ａ ＧＴＣＴＧＧＣＴＣＣＧ ＧＡＧ Ｇ Ａ 3 ,反义寡核苷酸为 5 Ｔ Ｃ ＣＴＣＣＧＧＡＧＣＣＡＧＡＣ Ｔ Ｔ 3 ( 为硫代磷酸修饰的碱基 ) ,由中科院上海细胞所合成和纯化。2 .细胞培养 :大鼠Ｃ6胶质瘤细胞培养于 1 0 %ＣＳＤＭＥＭ中 ,置 37℃、5 %ＣＯ2 培养箱中培养。3 .光镜 :细胞悬液接种于含无菌玻片的培养瓶中 ,培养 6ｈ。分组如下 :反义组 ,加入反义寡核苷酸1 0 μｍｏｌ/Ｌ ;正义组 ,加入正义寡核苷酸 1 0 μｍｏｌ/Ｌ ;空白组 ,…     

白藜芦醇对C6鼠胶质瘤细胞抑制生长和诱导凋亡的作用

白藜芦醇（resveratrol，Res）是一种广泛存在于植物中的植物补体，Res不但可以作为一种抗氧化剂，有防治心血管疾病的功效。而且具有抗炎和抗癌的作用，它可以在肿瘤的起始、发生、进展等各个阶段通过多种途径起到抑制肿瘤的作用。近年来有关胶质瘤的大量研究证实，表皮生长因子受体（epidermal growth factor receptor，EGFR）的基因扩增和过表达是恶性胶质瘤发生、发展的重要始动事件之一，其介导的信号转导通路发挥着关键的作用旧J。本实验进一步就Res对C6鼠脑胶质瘤细胞生长增殖的抑制、诱导细胞凋亡及其与胶质瘤增殖、凋亡相关的EGFR等重要基因变化进行了探讨。     

局部热化疗诱导鼠C6胶质瘤细胞凋亡的研究

目的研究局部热化疗对大鼠胶质瘤细胞凋亡的作用,探讨其作为胶质瘤辅助治疗的可行性。方法将传代培养的C6细胞接种于大鼠背部皮下熏肿瘤生长至1.5～2cm直径时,分组进行局部热疗、化疗和热化疗。继续观察皮下肿瘤生长情况,测量瘤体体积和重量;用免疫组化s-p法检测bax、bcl-2蛋白表达;用HE染色法、电镜、TUNEL法观察细胞凋亡;电镜观察肿瘤血管的变化。结果肿瘤接种32d后,热疗、化疗及热化疗组肿瘤的重量分别为(1.95±0.34)g,(1.86±0.40)g,(1.09±0.21)g,明显较对照组(2.95±0.36)g轻(P<0.001);各治疗组肿瘤体积也明显小于对照组;各治疗组bax蛋白表达亦明显强于对照组(P<0.001),且热化疗组也强于单纯热疗和化疗组(P<0.001);bcl-2蛋白表达改变不明显(P>0.05);热疗、化疗及热化疗组细胞凋亡指数分别为18.96±2.54、20.05±3.64、36.62±8.96,明显大于对照组的4.68±1.68(P<0.001)。各治疗组肿瘤微血管外径变小,血管壁变厚,血管减少,血管内皮细胞紧密连接增多,细胞连接间隙减小,有的血管甚至只能发现完整的基膜而缺乏内皮细胞。结论①热疗、化疗能抑制肿瘤增殖,且阿霉素与热疗联合使用有协同作用。②热疗、化疗、热化疗能促进bax蛋白表达,使bcl-2/bax比值减小,诱导细胞凋亡。③热疗可直接损伤细胞和周围血管,     

X-刀治疗大鼠C6脑胶质瘤诱发的细胞凋亡

目的：观察X-刀治疗胶质瘤的作用，并探讨诱发细胞凋亡效应。方法：采用DNA琼脂糖凝胶电泳、超微结构观察及新兴的DNA片段末端标记方法对各组C6大鼠脑胶质瘤发生的细胞凋亡进行研究。结果：15、20及30Gy各组在治疗后24h内均出现明显的细胞凋亡，DNA末端标记法检测的结果不但与前述方法结果相符，还显示出其准确性高等特性。研究中还发现各治疗组均有坏死同时出现。结论：X-刀治疗后诱发细胞凋亡的同时还致细胞坏死，这种效应随剂量增加渐趋明显，构成SRS治疗胶质瘤发挥作用的两个方面。     

p16基因抑制脑胶质瘤细胞恶性增殖并诱导细胞凋亡

目的：探索p16基因在脑胶质瘤发生过程中的作用及其与脑胶质瘤细胞凋亡的关系。方法：采用脂质体,磷酸钙＋DMSO休克转染的方法,分别将外源野生型p16基因导入胶质瘤细胞株U251,SWO,观察p16基因短暂转染与长期稳定转染对胶质瘤细胞的作用,并筛选阳性克隆。同时以空载体质粒pCDNA3为对照,免疫组化,Northern杂交检测p16基因表达,对转染后细胞生长的抑制,细胞周期,凋亡及裸鼠致瘤能力的变化进行分析,结果：外源p16基因的高水平表达显著抑制了胶质瘤U251,SWO细胞的生长,克隆形成率减少,肿瘤细胞发生了G1期阻滞,p16基因短暂转染可诱导胶质瘤细胞凋亡,而长期稳定转染无明显的凋亡诱导作用。结论：外源野生型p16基因可抑制胶质瘤细胞恶性增殖并诱导细胞凋亡,肿瘤细胞的异质性特点与p16基因转染后的“自然选择”作用可能是p16基因长期稳定转染无明显凋亡诱导作用的主要机制。     

p53基因联合顺铂对实验性胶质瘤的治疗作用

目的观察p53基因和顺铂对实验性胶质瘤的治疗作用。方法先以人胶质瘤细胞株U251建立裸鼠胶质瘤模型,然后瘤体内注射p53基因和/或腹腔内注射顺铂,定期测量肿瘤的大小。结果观察30d,p53基因联合顺铂治疗组的肿瘤大小为(0.44±0.05)cm3,顺铂治疗组为(0.92±0.03)cm3,p53基因治疗组为(1.16±0.10)cm3,对照组为(1.72±0.17)cm3,各组间差异有显著性意义(P<0.05)。结论对实验性胶质瘤,p53基因联合顺铂治疗组的疗效好于单用p53基因或顺铂治疗组。     

Growth inhibition and induction of differentiation by panaxydol in rat C6 glioma cells

OBJECTIVES: Panaxydol is a naturally occurring non-peptidyl small molecule isolated from the lipophilic fractions of Panax notoginseng, a well-known Chinese traditional medicine. In this study, we aimed to investigate the effects of panaxydol on growth inhibition and its mechanisms in C6 rat glioma cells. METHODS: The effects of panaxydol on cell proliferation, morphologic changes, glial fibrillary acidic protein (GFAP) expression and cell cycle regulation in rat C6 cells were evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, hematoxylin and eosin (HE) staining, immunocytochemistry, flow cytometric analysis and Western blot respectively. RESULTS: Panaxydol markedly inhibited the proliferation of C6 cells in a dose-dependent manner with IC50 of 39.5 +/- 2.3 microM. In addition, the cell morphologic changes and increased expression of GFAP in C6 cells in the presence of panaxydol implied a cellular differentiation. Flow cytometric analysis revealed that panaxydol-treated cells accumulated in G0/G1 phase with a marked decrease in the number of C6 cells at S phase. Western blot analysis demonstrated that panaxydol resulted in an increase in the protein expression of p27 in C6 cells as early as 3 hours after treatment consistent with the differentiation response, but protein expression of p53, p21, p16 and pRb remained unchanged. CONCLUSION: These findings suggest that panaxydol inhibits the proliferation of C6 cells via G0/G1 cell cycle arrest in association with induction of p27 expression and differentiation.     

Ethanol inhibits G-protein-mediated glucose uptake by C6 glioma cells

The mechanism of ethanol inhibition of glucose uptake was investigated using C6 glioma cells. Basal [3H]2-deoxy-D-glucose (2DG) uptake by C6 cells was inhibited by ethanol in a concentration-dependent manner. Fifty, 75 and 100 mM ethanol significantly inhibited basal 2DG uptake by 12, 20 and 23%, respectively (p < 0.05). Carbachol (an agonist acting via G protein-coupled receptors) stimulated the uptake by 26% (p < 0.05). In the presence of 100 mM ethanol, the ability of carbachol to stimulate 2DG uptake was abolished. In contrast, ethanol did not inhibit the ability of insulin to stimulate 2DG uptake. These results suggest that ethanol inhibits 2DG uptake by selectively interfering with G protein-mediated signal transduction pathway.     

Lithium chloride inhibits thrombin-induced intracellular calcium mobilization in C6 rat glioma cells

In this study, the authors have demonstrated the effect of lithium, a typical mood stabilizer, on thrombin-evoked Ca2+ mobilization in C6 cells to elucidate the action mechanisms of the drug. Thrombin-induced Ca2 mobilization was reduced 24 hr after 1 or 10 mM lithium chloride (LiCl) pretreatment. The Ca2+ rise was reduced in a time-dependent manner, and the significant inhibition was observed 9 hr pretreatment with 10 mM LiCl. On the other hand, pretreatment of the cells with 10 mM LiCl for 24 hr did not alter the amount of Galphaq/11 significantly. Pretreatment with 10 mM LiCl for 24 hr failed to reduce the 5-HT-induced Ca2+ mobilization or to affect the desensitization of the 5-HT signal. Finally, thrombin-elicited Ca2+ rise was markedly inhibited in the presence of 0.05 U/ml plasmin, however, the Ca2+ rise was not further attenuated in the presence of plasmin in C6 cells pretreated with LiCl for 24 hr. These results indicate that pretreatment with LiCl attenuated thrombin-evoked intracellular Ca2+ mobilization in plasmin sensitive manner in C6 rat glioma cells. Thus, it is important to investigate the effect of lithium on thrombin-induced cellular responses to clarify the action mechanism of lithium in relation to some abnormality in thrombin-evoked Ca2+ rise observed in bipolar disorders.     

苯乙酸对胶质瘤C6细胞增殖和凋亡的影响

目的观察诱导分化剂苯乙酸（PA）对胶质瘤C6细胞增殖和凋亡的影响。方法体外培养胶质瘤C6细胞，用0、2．5、5．0和7．5mmol／L不同浓度的PA，分别在PA诱导24、48、72、96h后。甲基噻唑基四唑（MTT）比色法检测细胞增殖抑制率，进一步用免疫细胞化学检测胶质纤维酸性蛋白（GFAP）的表达变化，末端脱氧核糖核酸转移酶介导的脱氧尿嘧啶核苷酸缺口末端标记（TUNEL）检测细胞凋亡。结果PA显著抑制c6细胞增殖，随药物浓度的增加和作用时间的延长，细胞增殖抑制率增加。PA作用后GFAP表达增强。随PA浓度和作用时间的增加，细胞凋亡率增加。结论PA对胶质瘤C6细胞有显著的增殖抑制和诱导凋亡作用，呈时间剂量依赖性。     

Dehydroepiandrosterone inhibits the death of immunostimulated rat C6 glioma cells deprived of glucose

Pretreatment of interferon-gamma and lipopolysaccharides made C6 glioma cells highly vulnerable to glucose deprivation. Neither 12 h of glucose deprivation nor 2-day treatment with interferon-gamma (100 U/ml) and lipopolysaccharides (1 microg/ml) altered the viability of C6 glioma cells. However, significant death of immunostimulated C6 glioma cells was observed after 5 h of glucose deprivation. The augmented death was prevented by dehydroepiandrosterone (DHEA) treatment during immunostimulation, but not by DHEA treatment during glucose deprivation. DHEA reduced the rise in nitrotyrosine immunoreactivity, a marker of peroxynitrite, and superoxide production in glucose-deprived immunostimulated C6 glioma cells. DHEA, however, did not protect glucose-deprived C6 glioma cells from the exogenously produced peroxynitrite by 3-morpholinosydnonimine. Further, DHEA did not alter the production of total reactive oxygen species and nitric oxide in immunostimulated C6 glioma cells. Superoxide dismutase (SOD) and the synthetic SOD mimetic Mn(III)tetrakis (4-benzoic acid) porphyrin inhibited the death of glucose-deprived immunostimulated C6 glioma cells. In addition, a superoxide anion generator paraquat reversed the protective effect of DHEA on the augmented death. The data indicate that DHEA prevents the glucose deprivation-evoked augmented death by inhibiting the production of superoxide anion in immunostimulated C6 glioma cells.     

Regulation of cAMP levels by protein kinase C in C6 rat glioma cells

Cultures of rat C6 rat glioma cells exhibit a diminished response to isoproterenol and forskolin after being treated with phorbol 12,13-dibutyrate (PDbU). An IC50 for PDbU of 38 +/- 5 nM and 62 +/- 8 nM was observed in the isoproterenol and forskolin response, respectively. Similarly, C6 cultures exhibited a diminished response to isoproterenol and forskolin after an overnight incubation with phospholipase C. We previously demonstrated that this treatment will increase diacylglycerol levels in these cells (Bressler: J Neurochem 48:181-186, 1987). An IC50 for phospholipase C of 6.0 +/- 0.1 x 10(-1) and 7.0 +/- 0.1 x 10(-1) units/ml was observed for the isoproterenol and forskolin response, respectively. A kinetic analysis suggests that the site of PDbU-mediated inhibition to beta-adrenergic and forskolin stimulation was different. Degradation of cAMP was a contributory factor since elevated cAMP levels decreased faster in PDbU treated cells than in nontreated cells. In addition, PDbU treated cells exhibited a significantly higher level of phosphodiesterase activity. We conclude that activation of protein kinase C and subsequent stimulation of phosphodiesterase activity contributes to the inhibition of the beta-adrenergic and forskolin mediated increase in cAMP levels in intact C6 rat glioma cells. The consequences of lower cAMP levels in sustaining differentiated function in the C6 rat glioma cell line will be discussed.     

Calcium-mediated endothelin signaling in C6 rat glioma cells

Endothelin induced intracellular Ca(2+)signaling was studied in C6 rat glial cells. Endothelins 1 and 3 increased transiently intracellular Ca(2+)concentration, endothelin 3 being less potent inducer. Dibutyryl-cAMP treated cells responded with less sensitivity. While BQ123, a specific endothelin A receptor antagonist, inhibited both endothelins induced response in proliferating cells, it failed to inhibit in dibutyryl-cAMP treated ones. IRL1620, a specific endothelin B receptor agonist, was devoid of any significant effect. Although re-stimulation by both endothelins after endothelin-1 did not cause any Ca(2+)oscillation, both endothelins evoked new Ca(2+)transient after endothelin-3 stimulation. Our findings suggest that endothelin induced Ca(2+)signaling is mediated probably through the receptor A in proliferating C6 cells. The lack of both BQ123 and IRL 1620 effect in dibutyryl-cAMP treated cells could be caused by an alteration of endothelin A receptor alone, by a change of receptor expression pattern, or by more complex postreceptor mechanism.     

Induction of GDNF mRNA expression by melatonin in rat C6 glioma cells

In order to determine the physiological effect of melatonin on glial cell line-derived neurotrophic factor (GDNF), which is reportedly up-regulated by high doses of this hormone, concentration-dependent studies were carried out in cultured cells. RT-PCR studies indicated that, in addition to GDNF, rat C6 glioma cells express both of the G protein-coupled melatonin receptor subtypes, MT1 and MT2. When C6 cells were treated with physiological (0.05-1 nM) or higher (10 and 100 nM) concentrations of melatonin for 24 h, a significant induction of relative GDNF mRNA levels (n = 4) was detected by semi-quantitative RT-PCR. These findings suggest that induction of GDNF is involved in physiological neuroprotection by melatonin. Given the potency of GDNF in maintaining nigrostriatal dopaminergic integrity, understanding the mechanisms of its induction by melatonin could provide novel therapies for Parkinson's disease.     

ATRA诱导胶质瘤C6细胞凋亡过程中Bcl-2和Caspase-3表达的研究

目的研究全反式维甲酸（ATRA）诱导胶质瘤C6细胞凋亡过程中Caspase-3和Bcl-2表达情况,探讨ATRA诱导胶质瘤C6细胞凋亡的机制。方法用四甲基偶氮唑蓝（MTT）比色法绘制ATRA不同浓度不同时间（6、12、24、48、72h）对胶质瘤C6细胞作用后细胞生长曲线,逆转录酶-多聚酶链反应（RT—PCR）检测凋亡相关基因Bcl-2和Caspase-3 mRNA水平的表达变化影响,Western blot分析Caspased和Bcl-2在胶质瘤C6细胞中的表达情况。结果胶质瘤C6细胞经ATRA诱导后,细胞的增殖明显受到抑制,通过RT-PCR结果证实Bcl-2 mRNA在ATRA作用下明显呈低表达而Caspase-3 mRNA的表达随时间延长逐渐增高,Western blot检测结果显示ATRA作用后,下调凋亡抑制蛋白Bcl-2的表达并激活了凋亡蛋白Caspase-3的表达。结论ATRA对胶质瘤C6细胞的增殖具有抑制作用。     

苯乙酸对胶质瘤C6细胞凋亡和细胞内游离Ca2+浓度的影响

目的观察苯乙酸(PA)对胶质瘤C6细胞凋亡和细胞内游离Ca2 浓度的影响,探讨其作用机制。方法体外培养胶质瘤C6细胞,PA诱导分化后,用透射电镜和Annexin V/PI标记流式细胞仪检测细胞凋亡,Fluo-3/AM探针激光共聚焦显微镜检测细胞内游离Ca2 浓度的变化。结果电镜观察到凋亡细胞,PA对C6细胞早期凋亡率无影响,主要增加晚期凋亡率,随PA浓度的增加而增加。PA能明显增加细胞内游离Ca2 浓度,并且随药物浓度的增加而增加。结论PA能诱导C6细胞凋亡,呈剂量依赖性,PA诱导C6细胞凋亡的机制可能与增加细胞内游离Ca2 浓度有关。