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。     

VEGF抗体治疗胶质瘤的实验研究

目的 以血管内皮生长因子（ｖａｓｃｕｌａｒ ｅｎｄｏｔｈｅｌｉａｌ ｇｒｏｗｔｈｆａｃｔｏｒ,ＶＥＧＦ）抗体进行抗血管生成治疗胶原瘤的研究。方法 应用ＶＥＧＦ抗体分别作用于Ｃ６胶原瘤细胞系的体外培养细胞和动物模型,计算细胞存活率及抑溜率,ＡＢＣ免疫组化技术检测体内生长胶质瘤中微血管密度。结果 ＶＥＧＦ抗体不抑制体外培养细胞生长,细胞存活率为１００％。对体内生长的胶质瘤则有显著抑制作用且呈剂量依赖关     

EGCG对肿瘤生物抑制机制影响的探讨

目的:探讨表没食子儿茶素没食子酸酯(EGCG)通过抑制血管内皮生长因子(VEGF)的血管生成效应减少肿瘤生长和血管生成。方法:MTT法检测内皮细胞生长、Transwell检测内皮细胞迁移,同时检测内皮细胞体外小管形成情况及Matrigel胶塞体内实验检测体内血管生成情况;建立异位胃癌裸鼠模型,检测肿瘤生长及肿瘤组织微血管密度,明确EGCG对肿瘤生长和血管生成的抑制作用及其机制。结果:体外实验显示,随着EGCG处理时间和剂量的增加,VEGF诱导生长的内皮细胞数呈时间和剂量依赖性地减少;随着EGCG剂量的增加,VEGF诱导迁移的内皮细胞数和形成的小管样结构也剂量依赖性地减少,差异有统计学意义,P<0.05;Matrigel胶塞体内实验也显示EGCG抑制VEGF诱导的胶塞血管化;动物实验显示治疗组肿瘤生长缓慢,生长曲线明显低于对照组,平均肿瘤抑制率为60.4%,P<0.01;治疗组肿瘤组织微血管密度显著降低,P<0.01。结论:EGCG可以抑制VEGF诱导的血管生成,从而抑制肿瘤生长和血管生成。     

EGCG对肿瘤生物抑制机制影响的探讨

目的：探讨表没食子儿茶素没食子酸酯（EGCG）通过押制血管内皮生长因子（VEGF）的血管生成效应减少肿瘤生长和血管生成。方法：MTT法检测内皮细胞生长、Transwell检测内皮细胞迁移，同时检测内皮细胞体外小管形成情况及Matrigel胶塞体内实验检测体内血管生成情况；建立异位胃癌裸鼠模型，检测肿瘤生长及肿瘤组织微血管密度，明确EGCG对肿瘤生长和血管生成的抑制作用及其机制。结果：体外实验显示，随着EGCG处理时间和剂量的增加，VEGF诱导生长的内皮细胞教呈时间和剂量依赖性地减少；随着EGCG剂量的增加，VEGF诱导迁移的内皮细胞数和形成的小管样结构也剂量依赖性地减少，差异有统计学意义，P〈0．05；Matrigel胶塞体内实验也显示EGCG抑制VEGF诱导的胶塞血管化；动物实验显示治疗组肿瘤生长缓慢，生长曲线明显低于对照组，平均肿瘤抑制率为60．4％，P〈0．01；治疗组肿瘤组织微血管密度显著降低，P〈0．01。结论：EGCG可以抑制VEGF诱导的血管生成，从而抑制肿瘤生长和血管生成。     

血管抑素抑制人脑胶质瘤生长及病理学研究

目的：观察人血管抑素（Angiostatin, AS）对小鼠脑胶质瘤皮下移植瘤生长的抑制作用。方法：用MTT法观察AS对血管内皮细胞系ECV304和人脑胶质瘤细胞系TJ905增殖的影响；建立荷G422脑胶质瘤小鼠皮下移植模型，皮下注射AS，计算瘤体比和抑瘤率。结果： (1)AS对ECV304的抑制作用随着剂量的增加而增强，AS对TJ905无影响；(2)动物实验显示，AS剂量为10 mg·kg-1·d-1和50 mg·kg-1·d-1时抑瘤率分别为21.8%和84.3%；(3)免疫组化Ⅷ因子染色表明实验组与对照组之间在血管发育及数量方面均有差别。结论： AS在体外对胶质瘤细胞无抑制作用，在体内能通过抑制血管内皮细胞增殖而抑制胶质瘤的生长。     

光激活金丝桃素对大鼠C6胶质瘤抑制作用及其对bFGF和bFGF-R及MVD的影响

目的:研究金丝桃素被光激活后对C6胶质瘤的抑制作用及其对bFGF、bFGF-R和微血管密度(microvesseldensity,MVD)的影响。方法:将接种C6胶质瘤细胞后的40只Wistar大鼠随机分为5组,空白对照组、高和低剂量金丝桃素作用组、高和低剂量金丝桃素 光照组,16d后切取肿瘤组织,称质量计算抑瘤率,标本用SP法免疫组化染色,检测瘤组织中bFGF、bFGF-R和MVD。结果:1)光照下,当金丝桃素为0·8μg/mL时,抑瘤率为67·2%,金丝桃素为2·0μg/mL时抑瘤率为82·4%,非光照金丝桃素组对肿瘤无抑制作用。2)bFGF在各组大鼠C6胶质瘤组织中均有表达,表达值较高,各组间差异无统计学意义。空白对照组中bFGF-R表达较高,且微血管计数最多。金丝桃素 光照组中bFGF-R蛋白表达和MVD明显低于非光照组及对照组,P=0·019,P=0·007,呈剂量依赖关系。bFGF-R与肿瘤MVD呈正相关,rbFGF-R=0·85。结论:光激活金丝桃素对bFGF-R有抑制作用,对bFGF表达没有影响。光激活的金丝桃素能抑制大鼠C6胶质瘤生长的机制,可能是抑制肿瘤组织中bFGF-R后,阻止了bFGF发挥效应,抑制了肿瘤间质血管生成,而抑制肿瘤的生长。     

腺病毒介导的大鼠增殖抑制基因抑制大鼠胶质瘤的生长

目的:探讨腺病毒介导的大鼠增殖抑制基因(rat hyperplasia suppressor gene,rHSG)对大鼠胶质瘤生长的抑制作用.方法:建立大鼠C6胶质瘤动物模型,分别在接种C6胶质瘤细胞后第4和11天,于肿瘤原位注射0.9％氯化钠溶液(对照组)、重组腺病毒Adv-rHSG-GFP和腺病毒Adv-GFP,并于接种C6胶质瘤细胞后第9、15和21天取脑胶质瘤观察肿瘤的生长情况,免疫组织化学法检测增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)和rHSG蛋白的表达以及微血管密度(microvessel density,MVD),RT-PCR和蛋白质印迹法检测rHSG mRNA及蛋白的表达.结果:Adv-rHSG-GFP组肿瘤体积明显小于对照组和Adv-GFP组(P＜0.01).Adv-rHSG-GFP组PCNA标记指数和MVD均低于对照组和Adv-GFP组.Adv-rHSG-GFP组rHSG mRNA和蛋白的相对表达量均明显高于对照组和Adv-GFP组(P＜0.01).结论:Adv-rHSG-GFP能够抑制胶质瘤血管生成和肿瘤细胞增殖,对恶性胶质瘤的生长具有明显的抑制作用.     

Tamoxifen体内外抑制胶质瘤细胞生长研究

目的：探讨Tamoxifen在体内外对胶质瘤细胞的抑制作用及其机制；方法：Tamoxifen在体内外作用于C6大鼠胶质瘤细胞系，并用MTT法、TUNEL法、免疫组化及动态MRI来检测效果；结果：Tamoxifen能在体外明显抑制胶质瘤细胞生长，在体内抑制作用尚不确定；Tamoxifen可抑制PKC及IGF-1表达；结论：Tamoxifen抑制胶质瘤细胞生长的机制之一可能是抑制PKC及IGF-1活性。     

TAM对C6细胞生长与侵袭能力的影响

目的:通过观察TAM对体外培养C6细胞生长状况、迁移能力与MMP-2蛋白表达的影响,为TAM用于胶质瘤的治疗提供理论依据。方法:观察不同剂量TAM对大鼠C6胶质瘤细胞生长曲线、细胞倍增时间及细胞形态的影响,研究TAM对C6细胞的生长抑制作用;细胞划痕法检测不同剂量TAM对C6细胞迁移能力的影响;FITC标记的可激活穿膜肽检测不同剂量TAM对C6细胞MMP-2蛋白表达的影响。结果:TAM显著抑制C6胶质瘤细胞的生长,且存在剂量、时间依赖关系;TAM显著抑制C6细胞迁移,存在剂量依赖关系;TAM对C6细胞活性MMP-2蛋白的表达未见明显影响。结论:虽然TAM不能抑制C6细胞MMP-2蛋白的表达,但可显著抑制其生长与迁移,因此有望用于胶质瘤的化学治疗。     

Roquinimex治疗人大肠癌的抗血管生成作用研究

目的：观察Roquinimex对人大肠癌细胞系SW1116的体内抑瘤效应,并探讨其可能的作用机制。方法：建立人大肠癌裸鼠移植瘤模型,比较各剂量Roquinimex的抑瘤效应的强弱。同时运用免疫组化方法,检测人大肠癌裸鼠移植瘤块的瘤灶内微血管密度（MVD）,并观察Roquinimex对鸡胚尿膜囊血管生长的影响。结果：体内实验显示Roquinimex能明显抑制SW1116裸鼠移植瘤的生长,各剂量Roquinimex治疗组的瘤块平均体积均小于空白对照组（P＜0．01）。各剂量Roquinimex治疗组瘤块微血管密度明显低于未经治疗组（P＜0．05）。在鸡胚尿膜囊血管生长模型中,Roquinimex明显抑制了鸡胚尿膜囊血管的生长。结论：Roquinimex能明显降低瘤块内微血管密度,抑制大鼠癌裸鼠移植瘤的体内生长,是一种有效的血管生长抑制剂。     

三苯氧胺对胶质瘤C6细胞垂体瘤转化基因表达及肿瘤生长影响的体内研究

目的 探讨体内三苯氧胺对胶质瘤C6细胞垂体瘤转化基因(PTTG)表达及细胞生长的影响.方法 建立SD大鼠C6胶质瘤移植动物模型,将32只荷瘤裸鼠随机分为4组:空白对照组、高(2 mg·kg-1·d-1)、中(0.2 mg·kg-1·d-1)、低(0.02 mg·kg-1·d-1)剂量三苯氧胺作用组,共给药20 d.空白对照组给等量的生理盐水.定期观察肿瘤生长情况,测量肿瘤体积,计算抑瘤率.处死全部动物模型,剔出肿瘤,逆转录-聚合酶链反应(RT-PCR)检测FITG mRNA表达.结果 与空白对照组相比,各组三苯氧胺均能抑制肿瘤生长,其体积抑瘤率分别为47.6%、35.5%、21.2%,各组间差异均有统计学意义(P<0.05);FITG mRNA的表达在低、中、高剂量组均降低,各组间差异均有统计学意义(P<0.05).结论 三苯氧胺能以量效的方式抑制胶质瘤PTTG的表达和肿瘤生长,本研究为临床应用三苯氧胺治疗胶质瘤提供了理论基础.     

表皮生长因子对胶质瘤C6细胞垂体瘤转化基因影响的实验研究

目的探讨表皮生长因子（EGF）在体外对胶质瘤C6细胞垂体瘤转化基因（PTTG）表达的影响。方法不同浓度的EGF（10ng／mL、20ng／mL和30ng／mL）在体外作用于胶质瘤C6细胞,半定量逆转录聚合酶链式反应（RT—PCR）检测PTTG mRNA表达,Western检测其PTTG蛋白表达。结果与空白对照组比较,RT—PCR和Western检测结果显示PTTG mRNA及其蛋白的表达在各EGF作用组均显著增高,各组间比较差异有统计学意义（P〈0.01）。结论EGF可以量效的方式上调PTTG的表达。     

Thrombin has a bimodal effect on glioma cell growth.

Using rat glioma C6 cells as a model, we have found a bimodal effect of alpha-thrombin on cell growth. In C6 cells treated with alpha-thrombin at concentrations from 0.02 nM to 1.0 nM, inhibition of cell proliferation was noted. Because the thrombin receptor agonist peptide TRAP-6 also induced inhibition of cell proliferation and the thrombin receptor antagonist peptide T1 prevented the inhibitory effect of alpha-thrombin on C6 glioma cell growth, thrombin receptor involvement in antiproliferative action of alpha-thrombin in C6 glioma cells is highly likely. However, stimulation of cell proliferation observed when C6 cells were treated with alpha-thrombin at higher doses (> 1.0 nM) seems to be mediated by as yet undefined thrombin receptor-independent biochemical mechanisms.     

Irradiated C6 glioma cells induce angiogenesis in vivo and activate endothelial cells in vitro

Malignant gliomas are angiogenesis dependent and present a remarkable degree of resistance to radiotherapy. In the present work, we studied the effect of irradiation of C6 glioma cells on their proliferation and activation in vitro and on glioma cell-induced angiogenesis in vivo and in vitro. Irradiation of C6 glioma cells decreased cell proliferation in a dose-dependent manner. Interestingly, metalloproteinase-2 and -9 expression and secretion, as well as integrin alpha(v) expression, increased with elevated doses of X rays 48 hr after irradiation and was mostly evident at the higher doses used. When pre-irradiated C6 cells were implanted on nonirradiated chicken embryo chorioallantoic membranes (CAMs), there was a significant dose-dependent increase in tumor induced angiogenesis, compared to angiogenesis induced by nonirradiated cells. Similar results were obtained when C6 cells were irradiated 48 hr after their inoculation onto nonirradiated CAMs. In the same line, conditioned medium from irradiated C6 cells significantly increased endothelial cell proliferation and migration in vitro, in a manner dependent on the dose of X rays. These results explain at least in part the low effectiveness of radiation therapy of malignant gliomas and support the notion that inhibition of angiogenesis in parallel with radiotherapy may represent a new therapeutic approach.     

The effect of calphostin C,a potent photodependent protein kinase C inhibitor,on the proliferation of glioma cells in vitro

Recent studies have suggested that the proliferation of malignant gliomas may result from activation of protein kinase C (PKC)-mediated pathways; conversely, inhibitionof PKC may provide a strategy for blocking tumor growth.In the current studies, we examined the effect of a novelPKC inhibitor, calphostin C, which is a selective, highlypotent, photo-activatable inhibitor of the PKC regulatorydomain, on the proliferation and viability of three established and three low-passage malignant glioma cell lines, four low-passage low-grade glioma cell lines, and in adult human and neonatal rat non-neoplastic astrocyte cell lines in vitro. Under light-treated conditions, calphostin C consistently inhibited cell proliferation in each of the tumor cell lines and in the neonatal rat astrocyte cell line with a 50% effective concentration of 30 to 50 ng/ml (40 to 60 nm), which was comparable to the previously reported median inhibitory concentration (IC₅₀) for PKC inhibition by calphostin C. Complete elimination of proliferation was achieved atconcentrations of 50 to 100 ng/ml (60 to 125 nM). Cell viability decreased sharply with calphostin C concentrations of 100 to 300 ng/ml (125 to 380 nM). In contrast, under light-shielded conditions, calphostin C had a comparatively modest effect on cell proliferation and viability, with a median effective concentration of approximately 300 ng/ml. No significant inhibition of proliferationwas noted in the non-neoplastic adult astrocyte cell line under either light-treated or light-shielded conditions. These findings provide further evidence that PKC may play an essential role in mediating the proliferation of both benign and malignant glioma cells in vitro and may also contribute to the proliferation of non-neoplastic immature astrocytes. Light-sensitive inhibition of proliferation and viability by agents such as calphostin C may provide a novel strategy for applying photodynamic therapy to the treatment of neoplastic glial cells.     

Nerve Growth Factor Plays a Divergent Role in Mediating Growth of Rat C6 Glioma Cells via Binding to the P75 Neurotrophin Receptor

Dysregulation of proliferation, differentiation and cell death play a major role in glial tumors, and there is evidence for regulatory mechanisms involving nerve growth factor (NGF) and its receptors in various CNS-derived tumor cell lines. The aim of our study was to observe the effect of exogenous recombinant NGF on C6 rat glioma growth, to characterize the role of endogenous NGF and the p75 neurotrophin receptor (p75) and to rule out whether p75 is necessary to mediate the effect of exogenous NGF. Recombinant exogenous NGF (1–100ng/ml) was applied under different serum conditions (0%, 1%, 5%) and knockdown of endogenous NGF and p75 was achieved by lipid-mediated antisense oligonucleotide treatment. In presence of serum, NGF had a positive whereas in absence of serum NGF produced a negative effect on C6 cell number. A knockdown of NGF or p75 increased cell numbers and enhanced BrdU incorporation. In p75-knocked down cells NGF did not enhance C6 glioma growth in presence of serum. We conclude that (1) exogenous recombinant NGF enhances C6 glioma growth under serum conditions but decreases cell number in absence of serum, that (2) the effect of exogenous NGF is mediated by p75 alone or by heterodimers containing p75 and that (3) either basal levels of endogenous NGF or basal levels of p75 receptor moderate C6 glioma growth and represent an autoregulatory potential of C6 glioma cells.     

冬凌草甲素对C6大鼠脑胶质瘤裸鼠异种移植模型的抗肿瘤疗效研究

[目的]利用C6大鼠脑胶质瘤裸鼠异种移植模型研究冬凌草甲素对大鼠C6脑胶质瘤增殖的抑制效果。[方法]应用C6大鼠脑胶质瘤细胞株建立了该肿瘤的BALB／C裸鼠异种移植模型,腹腔注射低、中、高3个剂量的冬凌草甲素和固定剂量的对照化疗药物卡氮芥,比较给药前后的相对肿瘤增殖率、肿瘤抑制率和肿瘤体积变化．单因素方差分析比较各组间差异。[结果]在给药后的22d,冬凌草甲素低、中、高3个剂量组所对应动物模型的相对肿瘤增殖率均小于100％,且随着冬凌草甲素给药剂量的增加而降低;肿瘤抑制率均为正值,且随着给药剂量的增加而升高,但3个剂量组之间差异无统计学意义（F=7．59,P〉0．05）;在给药后的22d内,未给药组和所有给药组动物模型体内的肿瘤体积均呈逐日增大的趋势,但在12d后,高剂量冬凌草甲素给药组动物模型的肿瘤体积增量要显著小于未给药组（F=41．15,P〈0．05）,在给药组中,随着给药剂量的增加,在同一天动物模型所对应的肿瘤体积增量呈现递减趋势。[结论]冬凌草甲素对大鼠C6脑胶质瘤的增殖具有一定的抑制效果。     

The safety and efficacy of magnetic nano-iron hyperthermia therapy on rat brain glioma

Gliomas are a group of heterogeneous primary central nervous system tumors arising from glial cells. These tumors are associated with high morbidity and mortality. New opportunities for the development of effective therapies for malignant gliomas are urgently needed. Magnetic nano-particles can heat up tumor tissues and induce the killing of cancer cells. However, the in vivo action of magnetic nano-iron hyperthermia on brain gliomas has not been widely investigated. The safety, efficacy, and suitable dose of hyperthermia therapy remain unknown. We successfully established a rat model of brain glioma by injecting C6 glioma cells into the right caudate nuclei of rats. Fixed doses (2.5, 5, or 10 mg) of magnetic nano-iron were then injected into the tumors of tumor-bearing rats. The survival time of tumor-bearing rats was subsequently observed, and imaging studies were conducted on the brain tumors. Of the 80 rats that underwent C6 glioma cell implantation, 70 exhibited decreased mobility and appetite, and wasting. Establishment of this brain glioma model was confirmed to be successful by magnetic resonance imaging. After injection of different doses of magnetic nano-iron, the survival times of the different dose groups of tumor-bearing rats were not significantly different. However, the tumor size exhibited a significant decrease with magnetic nano-iron hyperthermia therapy. Injection of various doses of magnetic nano-iron was safe in tumor-bearing rats. The effective doses were 2.5 and 5 mg. Magnetic nano-iron hyperthermia significantly shrank the brain gliomas in tumor-bearing rats.     

Aurora kinase B/C inhibition impairs malignant glioma growth in vivo

Inhibition of Aurora kinase B has been evaluated as a therapy to block solid tumor growth in breast cancer, hepatocellular carcinoma, lung adenocarcinoma, and colorectal cancer models. Aurora kinase inhibitors are in early clinical trials for the treatment of leukemia. We hypothesized that Aurora B inhibition would reduce malignant glioma cell viability and result in impaired tumor growth in vivo. Aurora B expression is greater in cultured malignant glioma U251 cells compared to proliferating normal human astrocytes, and expression is maintained in U251 flank xenografts. Aurora B inhibition with AZD1152-HQPA blocked cell division in four different p53-mutant glioma cell lines (U251, T98G, U373, and U118). AZD1152-HQPA also inhibited Aurora C activation loop threonine autophosphorylation at the effective antiproliferative concentrations in vitro. Reduction in cell viability of U251 (p53(R273H)) cells was secondary to cytokinesis blockade and apoptosis induction following endoreplication. AZD1152-HQPA inhibited the growth of U251 tumor xenografts and resulted in an increase in tumor cell apoptosis both in vitro and in vivo. Subcutaneous administration of AZD1152-HQPA (25 mg/kg/day × 4 days; 2 cycles spaced 7 days apart) resulted in a prolongation in median survival after intracranial inoculation of U251 cells in mice (P = 0.025). This is the first demonstration that an Aurora kinase inhibitor can inhibit malignant glioma growth in vivo at drug doses that are clinically relevant.