大鼠皮层C6脑胶质瘤模型的建立

目的：为脑胶质瘤的研究提供实验基础，建立稳定可靠的动物模型。方法：借助于脑立体定位技术，在25只雄性Wistar大鼠脑S1区接种含有10g／L琼脂糖的C6细胞悬液。随机分成4个周组和1个自然死亡组后观察大鼠的生存期，并采用MRI、病理解剖、HE染色及GFAP免疫组织化学方法，动态观察各周组肿瘤生长情况。结果：在不同周组中，所有检查均显示颅内肿瘤形成，GFAP阳性。4周内无死亡，自然死亡组在第5周死亡2只、余3只于第6周死亡。结论：该方法建立的S1区脑胶质瘤模型具备与人脑胶质瘤生长相似的特性，肿瘤形成时间短，生存期稳定，且无脑外转移和颅外扩散，适合各种脑胶质瘤实验治疗研究。     

鼠脑胶质瘤模型制作方法的改良

 目的　建立简单易行、稳定的鼠C6脑胶质瘤模型。方法　SD鼠 2 0只 ,脑立体定向仪定向注射C6细胞至鼠脑尾状核 ,接种后 1、2、3、4周MRI测量肿瘤大小 ,每日观察鼠体重变化 ,处死后大体解剖和病理组织学检查。结果　MRI平扫见脑组织水肿明显 ,MRI增强可见肿瘤组织明显强化 ,中线移位 ,侧脑室受压或消失。颅内接种后星形细胞胶质瘤呈Ⅰ、Ⅱ、Ⅲ、Ⅳ级不等。结论　静脉注射套管针接种C6细胞 ,取材方便 ,简单易行 ,腹腔注射造影剂可获得较好的增强效果。     

土槿皮乙酸诱导大鼠神经胶质瘤C6细胞株凋亡

目的:研究土槿皮乙酸诱导大鼠神经胶质瘤C6细胞株凋亡及其机制.方法:采用MTT法检测0、0.625、1.25、2.5、5、10、20、40、80 μmol/L不同终浓度PAB对C6细胞增殖能力的影响,激光扫描共聚焦显微镜观察AO/EB荧光染色后细胞的凋亡情况,流式细胞术检测碘化丙啶(PI)染色后细胞周期的变化,免疫组化法测定C6细胞的凋亡相关蛋白Bcl-2的表达.结果:MTT结果显示,PAB呈时间剂量依赖性抑制C6细胞的生长.PAB处理24、48和72 h后,对C6细胞的半效致死剂量(IC50)分别为39.811、23.306和7.360 μmol/L.5.0、10.0、20.0 μmol/L PAB作用C6细胞72 h后的凋亡率分别为(50.5±0.7)%、(63.5±2.3)%和(80.0±1.2)%,与对照组(12.0±1.6)%比较均明显升高,差异有统计学意义(P<0.05).流式细胞仪检测结果显示5.0、10.0、20.0 μmol/L不同浓度PAB作用C6细胞72小时后,随着浓度的增加,G2/M期细胞逐渐增多,分别为(29.84±6.215)%,(34.58±1.187)%和(70.23±6.325)%,与对照组(7.79±0.570)%相比,后两组差异有统计学意义(P<0.05).C6细胞中Bcl-2蛋白阳性表达率随药物剂量的增大而减小.结论:PAB可抑制大鼠神经胶质瘤C6细胞增殖,阻滞细胞周期于G2/M期、下调Bcl-2表达为其可能的作用机制.     

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。     

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。     

大鼠皮层C6脑胶质瘤模型的建立

目的 :为脑胶质瘤的研究提供实验基础 ,建立稳定可靠的动物模型。方法 :借助于脑立体定位技术 ,在 2 5只雄性Wistar大鼠脑S1区接种含有 10g L琼脂糖的C6细胞悬液。随机分成 4个周组和 1个自然死亡组后观察大鼠的生存期 ,并采用MRI、病理解剖、HE染色及GFAP免疫组织化学方法 ,动态观察各周组肿瘤生长情况。结果 :在不同周组中 ,所有检查均显示颅内肿瘤形成 ,GFAP阳性。 4周内无死亡 ,自然死亡组在第 5周死亡 2只、余 3只于第 6周死亡。结论 :该方法建立的S1区脑胶质瘤模型具备与人脑胶质瘤生长相似的特性 ,肿瘤形成时间短 ,生存期稳定 ,且无脑外转移和颅外扩散 ,适合各种脑胶质瘤实验治疗研究。     

地昔帕明单用和合用替尼泊苷对大鼠脑胶质瘤C6细胞增殖的调控作用

目的：研究三环类抗抑郁药地昔帕明（ＤＭ）对脑胶质瘤Ｃ６细胞增殖的调控作用,并探讨与临床常用治疗脑瘤的化疗药物替尼泊苷（ＶＭ－２６）合用的协同效应。方法：采用ＭＴＴ比色法测定大鼠脑胶质瘤Ｃ６细胞增殖抑制作用和流式细胞术（ＦＣＭ）进行细胞周期分析。结果：ＤＭＩ（１０－８０μｍｏｌ／Ｌ）对Ｃ６细胞的增殖具有明显的抑制作用,呈浓度时间依赖关系,药物作用２４ｈ的ＩＣ５０（９５％置信区间）为２０．７（１７．３     

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。     

The pharmacokinetics of letrozole in brain and brain tumor in rats with orthotopically implanted C6 glioma, assessed using intracerebral microdialysis

Purpose

Emerging evidence suggests that primary and metastatic brain tumors may be sensitive to hormonal manipulations. However, the pharmacokinetics of compounds against such targets in the brain and, more importantly, in the brain tumor are not well characterized. Here, we investigated the pharmacokinetics of letrozole, a third-generation aromatase inhibitor, in the normal brain and in orthotopically implanted C6 glioma in Sprague–Dawley rats.

Methods

Intracerebral microdialysis was employed to determine the concentrations of unbound letrozole in the brain extracellular fluid (ECF) while simultaneously collecting blood samples (via jugular vein) to assess plasma levels of letrozole. Letrozole was administered intravenously at doses of 4, 6, 8 and 12 mg/kg, and ECF and blood samples were collected over 8 h. For assessing normal versus tumoral brain pharmacokinetics, letrozole (4 or 8 mg/Kg; i.v.) was administered 10 days after implantation of C6 glioma in the brain. Dual-probe intracerebral microdialysis was employed for assessing ECF samples from tumor-free and tumor-bearing regions of the brain.

Results

Normal brain ECF and plasma C _max and AUC_0–8h increased linearly with letrozole doses up to 8 mg/kg dose, but at 12 mg/kg, the pharmacokinetics were nonlinear. The relative brain distribution coefficients, AUC_ECF/AUC_{plasma (ub)}, were 0.3–0.98. The tumoral uptake of letrozole was 1.5- to 2-fold higher relative to tumor-free region.

Conclusions

Thus, letrozole permeability across the blood brain barrier is high, and the exposure to the brain is dose dependent. Furthermore, the brain tumoral letrozole levels are markedly higher than those in the tumor-free regions, which underscore potential selectivity of its activity against tumor cells.     

缓激肽对大鼠脑微血管内皮细胞和星形胶质细胞及胶质瘤细胞影响的体外研究

目的:体外探讨缓激肽(bradykinin,BK)的作用靶点细胞及BK选择性开放血脑屏障的可能机制。方法:通过免疫荧光测定脑血管内皮细胞、胶质细胞及C6细胞在不同剂量BK作用前后的细胞内钙离子变化,并且通过WesternBlot测定三组细胞的BKB2受体表达水平。结果:小剂量BK可以引起肿瘤细胞内的钙离子水平升高,而只有大剂量BK才能触发星形胶质细胞内的钙离子水平变化,脑血管内皮细胞对大、小剂量BK均无任何反应;三组细胞BKB2受体的WesternBlot整合密度值(integrateddensityvalue,IDV)分别为5000.12±1110.21(n=2)、18480.88±4119.86(n=3)和63032.13±2802.71(n=4),组间比较差异有统计学意义。结论:BK的直接作用靶点是星形胶质细胞及肿瘤细胞,两种细胞B2受体表达水平差异是小剂量BK选择性开放血肿瘤屏障的物质基础;BK可能通过某些细胞间信使起到调节脑血管内皮细胞通透性的作用。     

Pimonidazole binding in C6 rat brain glioma: relation with lipid droplet detection

In C6 rat brain glioma, we have investigated the relation between hypoxia and the presence of lipid droplets in the cytoplasm of viable cells adjacent to necrosis. For this purpose, rats were stereotaxically implanted with C6 cells. Experiments were carried out by the end of the tumour development. A multifluorescence staining protocol combined with digital image analysis was used to quantitatively study the spatial distribution of hypoxic cells (pimonidazole), blood perfusion (Hoechst 33342), total vascular bed (collagen type IV) and lipid droplets (Red Oil) in single frozen sections. All tumours (n=6) showed necrosis, pimonidazole binding and lipid droplets. Pimonidazole binding occurred at a mean distance of 114 microm from perfused vessels mainly around necrosis. Lipid droplets were principally located in the necrotic tissue. Some smaller droplets were also observed in part of the pimonidazole-binding cells surrounding necrosis. Hence, lipid droplets appeared only in hypoxic cells adjacent to necrosis, at an approximate distance of 181 microm from perfused vessels. In conclusion, our results show that severe hypoxic cells accumulated small lipid droplets. However, a 100% colocalisation of hypoxia and lipid droplets does not exist. Thus, lipid droplets cannot be considered as a surrogate marker of hypoxia, but rather of severe, prenecrotic hypoxia.     

Intrathecal treatment of C6 glioma leptomeningeal metastasis in Wistar rats with interlenkin-2

Summary The efficacy of intrathecal treatment of leptomeningeal metastasis (LM) with interleukin-2 (IL-2) was evaluated in an animal model using Wistar rats inoculated intracisternally with 10⁷ C6 glioma cells. Prior to the in vivo experiments the antiproliferative effects of human IL-2, and of murine IFN- and TNF- which are cytokines induced by IL-2 were tested in a colony forming assay. Only IFN- caused a dose-dependent inhibition of colony formation. Twelve animals were treated intracisternally with either 10⁵ IU IL-2 or control medium on day 0, 2, and 5 after tumor cell inoculation. Both IL-2 treated and sham-treated animals developed LM with a symptom-free survival of 7 to 9 days. There was no significant difference between treated and untreated animals regarding time to onset of symptoms and pattern of tumor growth. Infiltration of the tumor tissue with ED-1+ monocytes and macrophages, and CD8+ lymphocytes, however, was slightly increased in IL-2 treated animals. In a second experiment 4 non tumor-bearing Wistar rats were intracisternally injected with a single dose of 105 IU IL-2. These animals also showed slightly enhanced leptomeningeal infiltration with CD8+ lymphocytes compared to controls. We conclude that intrathecal application of high-dose IL-2 although eliciting a slight immune reaction within the leptomeninges does not inhibit leptomeningeal tumor growth or prolong symptom-free survival in our animal model of LM. These results raise doubt about the clinical efficacy of intrathecal IL-2 treatment in patients with LM.     

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。     

连接蛋白43基因治疗鼠C6脑胶质瘤的体内实验研究

目的 研究连接蛋白（Cx）43基因抑制脑胶质瘤生长的作用。方法 将Cx43基因表达缺失的大鼠C6胶质瘤细胞（对照组）和转染Cx43 cDNA的C6细胞（转染组）种植于SD大鼠右侧尾状核,荷载C6脑胶质瘤鼠用Cx 43 cDNA原位治疗（治疗组）,并以空载体治疗作为对照（空载组）,每组10只,观察各组大鼠一般情况、生存期、MRI动态变化及病理改变,通过原位杂交及免疫组化染色检测肿瘤Cx43 mRNA及蛋白表达,以AgNOR平均计数检测增殖活性,以Tunel法检测细胞凋亡。结果 对照组和空载组大鼠均于3周内死亡;转染组6只大鼠和治疗组8只大鼠观察120 d内无自然死亡。除治疗组1只尚有残瘤外,余瘤体消失,转染组和治疗组肿瘤细胞均有Cx43 mRNA及蛋白表达,且增殖治疗组1只尚有残瘤外,余瘤体消失。转染组和治疗组肿瘤细胞均有Cx43 mRNA及蛋白表达,且增殖活性下降,但凋亡不增加。结论 转染Cx43基因后的C6胶质瘤细胞致瘤性显著降低,且对荷载脑胶质瘤鼠具有明显的治疗作用,有可能成为恶性胶质瘤基因治疗的优先靶的之一。     

冷冻和rhTNF-α对C6大鼠脑胶质瘤的联合治疗作用

目的:探讨冷冻联合rhTNFα治疗C6大鼠脑胶质瘤的可行性。方法:借助于立体定位技术,将C6胶质瘤细胞接种于80只雄性Wistar大鼠脑S1区,待肿瘤长至第15天,直径约6mm时,随机将荷瘤鼠分为4组:G1、G2、G3、G4分别为生理盐水对照组、冷冻治疗组、rhTNFα治疗组及联合治疗组,每组20只,分别行相应的治疗。治疗后第15天,采用免疫组化检测PCNA的表达;行核磁共振(MRI)检查,测量肿瘤体积和抑瘤率。动态观察各组荷瘤鼠生存期和rhTNFα对荷瘤鼠的毒副作用。结果:联合治疗组PCNA的表达情况、肿瘤体积和生存期与其他各组相比有显著差异,且该组对肿瘤生长的抑制率(89.48%)明显高于冷冻组(66.31%)和TNFα组(49.01%),且大于理论抑瘤率(82.82%)。同时观察到rhTNFα对实验动物有一定毒副作用。结论:冷冻和rhTNFα联合应用,具有协同抗肿瘤作用,为进一步临床研究提供了实验依据。     

Modulation of cell growth and differentiation induced by prostaglandin D2 in the glioma cell line C6

Prostaglandins are produced mainly from arachidonic in various normal and neoplastic tissues and are well-known to be involved in the regulation of tumor cell proliferation, differentiation and metastasis. Prostaglandin D2 is cytotoxic to several human tumor cell lines, has antitumoral activity in vitro and is reported to be antimetastatic. However, the effects of prostaglandin D2 on tumoral cell differentiation have not been clarified. This paper studies the effects of prostaglandin D2 on C6 rat glioma cell differentiation and proliferation. Prostaglandin D2 administration in the culture medium is observed to alter cell morphology and cytoskeleton filament patterns. These alterations suggest a cell maturation, and correlate with a drop in proliferating capacity. The antitumoral activity of prostaglandin D2 involves a biomodulation of cell parameters which would affect neoplastic processes.     

125IUdR偶联c-myb反义寡核苷酸对大鼠C6脑胶质瘤细胞增殖的影响及其相关机制

目的:探讨125IUdR偶联c-myb反义寡核苷酸对C6脑胶质瘤细胞增殖和凋亡的影响及可能的机制。方法:60只大鼠随机余数法分成对照组、c-myb-ASODN处理组和125IUdR-ASODN处理组。MTT和TUNEL法检测各组大鼠肿瘤细胞增殖抑制率和凋亡率;免疫组化法检测Bax和Bcl-xl蛋白表达;RT-PCR法检测BaxmRNA和Bcl-xlmRNA含量;Western-blot法检测c-myb蛋白表达。结果:125IUdR-ASODN处理组C6胶质瘤细胞的增殖抑制率和细胞凋亡率(69·81%和11·03%)增高均较c-myb-ASODN处理组(40·37%和6·48%)增高,P均<0·01;与对照组凋亡率(0·95%)比较,差异有统计学意义,P均<0·01。c-myb-ASODN处理组和125IUdR-ASODN处理组C6胶质瘤细胞的Bax蛋白194·6±6·25增高及Bcl-xl蛋白59·0±3·56下降较ASODN组Bax蛋白147·4±5·23增高及Bcl-xl蛋白83·9±3·93下降,P均<0·01;125IUdR-ASODN和ASODN处理组BaxmRNA表达随着时间延长而显著升高,Bcl-xlmRNA及c-myb蛋白表达随着时间延长而显著下降,P均<0·01;且125IUdR-ASODN的作用明显强于c-myb-ASODN,P<0·01。结论:c-myb-ASODN和125IUdR-ASODN均具有促进C6胶质瘤细胞凋亡和抑制其增殖的作用,且125IUdR-ASODN的作用更为显著;c-myb-ASODN和125IUdR-ASODN可能通过Bax/Bcl-xl途径诱导C6胶质瘤细胞凋亡,其对Bax和Bcl-xl的调节发生在转录水平前的某一环节。