蛇毒解聚素在裸鼠胶质瘤模型中的干预治疗作用研究

目的研究蛇毒解聚素（CN）对裸鼠人脑胶质瘤动物模型的治疗可行性,探讨蛇毒解聚素抑制U87胶质瘤裸鼠移植瘤生长的作用机制。方法建立BALB／c裸鼠U87胶质瘤移植瘤模型,将荷瘤裸鼠随机分为2组,采用间质内注射给药方法。蛇毒解聚素按40μg每次给药。定期观察肿瘤生长情况,测量肿瘤体积,绘制肿瘤生长曲线并计算抑瘤率。全部BALB／c裸鼠移植瘤石蜡标本用SP法免疫组化染色,检测移植瘤组织中的微血管计数（MVD）,Ki-67标记指数以及碱性成纤维细胞生长因子（bFGF）。结果与溶媒对照组相比,蛇毒解聚素组均能抑制肿瘤生长（P〈0．01）,其体积抑瘤率为50％,并能明显降低肿瘤微血管密度、能下调移植瘤组织中bFGF的蛋白表达及Ki-67标记指数。结论蛇毒解聚素能明显抑制U87裸鼠移植瘤生长。     

白藜芦醇对裸鼠U87细胞移植瘤生长及血管生成的影响

目的 研究白藜芦醇(Res)对人脑胶质瘤系U87细胞的体内抗癌活性及血管生成的影响. 方法 BALB/c裸鼠20只,背部皮下接种胶质瘤细胞U87建立皮下移植瘤模型.随机数字表法分为10、100mg/kgRes治疗组,溶剂对照组,空白对照组,每组5只.观察4组裸鼠移植瘤的生长曲线并应用免疫组织化学法检测瘤组织中微血管密度(MVD)及血管内皮生长因子(VEGF)的表达:采用原位末端标记法(TUNEL)检测瘤组织细胞的凋亡. 结果与空白对照组及溶剂对照组相比,100 mg/kg Res治疗组裸鼠移植瘤的体积、重量降低;100 mg/kg Res治疗组瘤组织中MVD、VEGF的表达明显降低,凋亡细胞数增高,差异均有统计学意义(P<0.05). 结论 Res对U87人脑胶质瘤细胞裸鼠移植瘤的生长具有明显抑制作用,这可能与Res导致U87移植瘤细胞凋亡及血管生成减少有关.     

白藜芦醇对裸鼠U87细胞移植瘤生长及血管生成的影响

目的 研究白藜芦醇(Res)对人脑胶质瘤系U87细胞的体内抗癌活性及血管生成的影响. 方法 BALB/c裸鼠20只,背部皮下接种胶质瘤细胞U87建立皮下移植瘤模型.随机数字表法分为10、100mg/kgRes治疗组,溶剂对照组,空白对照组,每组5只.观察4组裸鼠移植瘤的生长曲线并应用免疫组织化学法检测瘤组织中微血管密度(MVD)及血管内皮生长因子(VEGF)的表达:采用原位末端标记法(TUNEL)检测瘤组织细胞的凋亡. 结果与空白对照组及溶剂对照组相比,100 mg/kg Res治疗组裸鼠移植瘤的体积、重量降低;100 mg/kg Res治疗组瘤组织中MVD、VEGF的表达明显降低,凋亡细胞数增高,差异均有统计学意义(P<0.05). 结论 Res对U87人脑胶质瘤细胞裸鼠移植瘤的生长具有明显抑制作用,这可能与Res导致U87移植瘤细胞凋亡及血管生成减少有关.     

人脑胶质瘤细胞裸鼠原位移植动物模型的建立

目的探讨人脑胶质瘤细胞裸鼠原位移植动物模型建立的技术方法。方法借助动物立体定向仪的引导,采用微注射方法将体外培养人脑胶质瘤细胞U87MG(悬浮于无血清RPMI 1640培养液中,细胞数为108/ml)接种于裸鼠(BALB/c)额叶白质区。接种后观察不同种实验鼠的生存状态,分别于接种后1 h至63 d的不同时间进行裸鼠脑肿瘤组织病理学检查和免疫组织化学分析。结果裸鼠脑内注射体外培养的人脑胶质瘤细胞U87MG的合适速率为0.05μl/min =1μl/20 min,细胞悬液体积1μl,细胞数105,注射时间20 min。按此方案注射U87MG细胞无沿针道返流,恰好在尾状核区形成近圆球形肿瘤体,成瘤率高(100%),肿瘤颅内生长稳定,组织病理学检查符合人脑胶质瘤形态特征,未见脑外转移。结论本研究的人脑胶质瘤细胞裸鼠原位移植动物模型建立方法精确可靠,重复性好。肿瘤符合人脑胶质瘤的生物学特性,该动物模型可作为研究人脑胶质瘤发生、生物学特性以及各种治疗评价的可靠动物模型。     

血管生成因子2在胶质瘤模型中的表达与shRNAs靶向治疗

目的 检验血管生成因子2(angiopoietin-2,Ang-2)在胶质瘤模型中的表达,应用RNA干扰技术靶向Ang-2,观察胶质瘤治疗效果.方法 建立荷瘤小鼠颅内U87胶质瘤模型,检测Ang-2和PCNA的表达;转染shRNAs表达质粒进入U87细胞,比较受试动物生存期,MRI观察肿瘤体积;脑组织行HE染色及免疫组化检测.结果 颅内模型中Ang-2在胶质瘤边缘的表达强于肿瘤内部(P<0.05),围绕于新生血管周围;靶向Ang-2的shRNAs基因治疗可延长受试动物生存期,降低新牟血管数量,但PCNA表达无统计学意义(P>0.05).结论 Ang-2主要表达于胶质瘤侵袭边缘,与肿瘤新生血管形成密切相关;靶向Ang-2的shRNAs技术可有效降低胶质瘤的侵袭能力.     

Single doublecortin gene therapy significantly reduces glioma tumor volume

We employed lentivirus‐based doublecortin (DCX), as a glioma suppressor gene therapy in an intracranial glioma tumor xenograft model in nude rats. Single DCX‐expressing lentivirus was directly administered into the tumor on day 8 after U87 tumor cell implantation. DCX treatment significantly reduced U87 glioma tumor volume (～60%) on day 14 after DCX lentivirus injection and significantly improved median survival of tumor‐bearing nude rats. DCX synthesis induced neuronal markers MAP2, TUJ1, and PSA‐NCAM and the glial marker glial fibrillary acidic protein (GFAP) in the implanted U87 glioma tumors. DCX synthesis induced GFAP that colocalized with tubulin in the mitotic stage, inhibited cleavage furrow during cytokinesis, and blocked mitosis in glioma cells. DCX lentivirus infection did not induce apoptosis but significantly inhibited expression of the proliferation marker Ki‐67 and the blood vessel marker von‐Willebrand factor (vWF). U87 and other glioma cells except for brain tumor stem cells (BTSCs) do not express neuronal markers or both neuronal and glial markers. DCX‐synthesizing glioma cells express a phenotype of antiangiogenic BTSC‐like cells with terminal differentiation that causes remission of glioma cells by blocking mitosis via a novel DCX/GFAP pathway. Direct local delivery of lentivirus‐based DCX gene therapy is a potential differentiation‐based therapeutic approach for the treatment of glioma. © 2009 Wiley‐Liss, Inc.     

Subclinical photodynamic therapy treatment modifies the brain microenvironment and promotes glioma growth

Photodynamic therapy (PDT) has been clinically investigated as an adjuvant local therapy for brain tumors. Therapeutic interventions intended to promote tumor cell death can also promote changes in the tumor microenvironment that could favor tumor growth. We have previously shown that PDT can activate pro-angiogenic factors in the normal rodent brain. This study seeks to further elucidate the effects of subtherapeutic doses of Photofrin-PDT on normal brain and to establish a mouse model for studying glioma progression in an environment modified by oxidative stress. Photofrin was administered to nude mice, and a defined intracranial area was illuminated with laser to deliver an optical dose equivalent to 80 J/cm(2). Three and 7 days after PDT, mice were sacrificed and brains were fixed and analyzed by immunohistochemistry. PDT treatment resulted in transient increase in cell proliferation, associated with a robust activation of astrocytes and microglia in the treated region, without causing substantial cell death. To test how this modified environment would affect glioma growth, human glioblastoma U87 cells were implanted in the PDT-treated hemisphere or in the control brain subjected to sham surgery. Significantly larger tumors were observed after 3 weeks in the PDT treated brains relative to control treatment. Our results indicate that subclinical Photofrin-PDT locally alters the brain homeostasis without inflicting significant disruption to the tissue architecture, providing a model to study the effects of the microenvironment on glioma growth, with implications for the optimization of the clinical use of PDT for brain tumors.     

白藜芦醇对裸鼠胶质瘤模型肿瘤生长的抑制作用

目的 探讨白藜芦醇（RES）不同途径给药对裸鼠胶质瘤模型肿瘤生长的抑制效果。方法 取80只雄性无胸腺裸鼠（BALB/c-nu;21~27日龄,体重10~12 g）,采用U87细胞建立人裸鼠脑胶质瘤原位移植模型,采用随机数字表法分为RES干预组（造模后10 d,开始给药,1次/d,40 mg/kg）和溶剂对照组（造模后10 d,开始给药,1次/d,10 mg/kg;溶剂为0.5%羧甲基纤维素钠）,根据给药途径,RES干预组又分为RES灌胃组和RES经鼻组,溶剂对照组又分为溶剂灌胃组和溶剂经鼻组,每组20只。经鼻给药采用经鼻滴入法。采用Kaplan-Meier法分析生存曲线;造模后14、21、28、35 d测定肿瘤体积;采用CD31标记胶质瘤血管内皮细胞并计算微血管密度（MVD）,采用免疫组化方法检测胶质瘤血管内皮生长因子（VEGF）以及Ki-67表达,采用TUNEL法检测胶质瘤细胞凋亡。结果 与溶剂对照组相比,RES干预组裸鼠生存时间明显延长（P<0.05）,造模后28、35 d肿瘤体积明显缩小（P<0.05）,肿瘤组织MVD明显减小（P<0.05）,肿瘤组织VEGF和Ki-67表达水平明显降低（P<0.05）,肿瘤细胞凋亡率明显增加（P<0.05）,而且,RES经鼻给药较灌胃给药作用更明显（P<0.05）。结论 RES可有效抑制裸鼠胶质瘤模型肿瘤生长,机制可能与抑制肿瘤血管生成和促进肿瘤细胞凋亡有关。与灌胃给药相比,经鼻给药肿瘤抑制效果更好。     

阿苯达唑抑制胶质瘤裸鼠模型肿瘤生长

目的 探讨阿苯达唑（ABZ）对胶质瘤裸鼠模型肿瘤生长的影响。方法 将术中获取的胶质母细胞组织消化为单细胞悬液用无血清干细胞培养基进行培养胶质母细胞瘤干细胞（GSC）,用含10%胎牛血清的DMEM培养基培养胶质瘤细胞系U87、U251、U172,以25、50、100、200 ng/ml的终浓度ABZ作用细胞,采用MTT法检测细胞增殖。右侧腋窝皮下注射0.2 ml（5×106个细胞）GSC及U87细胞悬液构建胶质瘤裸鼠模型,将30只胶质瘤裸鼠模型随机分为6组,GSC和U87细胞移植各3组,每种移植瘤模型均分为模型组（腹腔注射等体积DMSO）、低剂量ABZ组（腹腔注射ABZ,50 mg/kg）、高剂量ABZ组（腹腔注射ABZ,100 mg/kg）。定时测量肿瘤长径与短径,计算肿瘤体积;PCR法和免疫印迹法检测裸鼠肿瘤组织血管内皮生长因子（VEGF）mRNA和蛋白表达水平。结果 ABZ对GSC、U87、U251和A172细胞生长均具有明显抑制效果（P<0.05）,而且抑制效果具有浓度依赖性（P<0.05）,浓度>50 ng/ml抑制效果较好。腹腔注射ABZ后,高剂量和低剂量ABZ组移植瘤体积增长较对照组均明显减慢（P<0.05）。高剂量ABZ组和低剂量ABZ组肿瘤VEGF mRNA和蛋白表达水平较对照组均明显降低（P<0.05）。结论 ABZ可以抑制胶质瘤裸鼠模型肿瘤生长,可能与抑制肿瘤细胞增殖和血管生成有关。     

Curzerene suppresses progression of human glioblastoma through inhibition of glutathione S‐transferase A4

AimsGlioblastoma is the central nervous system tumor with the highest mortality rate, and the clinical effectiveness of chemotherapy is low. Curzerene can inhibit the progression of non‐small‐cell lung cancer, but its role in glioma has not been reported. The purpose of this study was to clarify the effect of curzerene on glioma progression and further explore its potential mechanism.MethodsThe expression of glutathione S‐transferase A4 (GSTA4) in glioblastoma and the effect of curzerene on the expression of GSTA4 and matrix metalloproteinase 9 and the activation of the mTOR pathway were detected by Western blotting and RT‐PCR, and the effects of curzerene treatment on glioma malignant character were detected by cell biological assays. The in vivo antitumor effects of curzerene were analyzed in a nude mouse xenograft model.ResultsCurzerene was found to inhibit the expression of GSTA4 mRNA and protein in U251 and U87 glioma cells, and this effect correlated with a downregulation of the proliferation of these cells in a time‐ and dose‐dependent manner. Invasion and migration were also inhibited, and curzerene treatment correlated with induction of apoptosis. Curzerene inhibited the activation of the mTOR pathway and the expression of matrix metalloproteinase 9, and it correlated with increased 4‐hydroxynonenal levels. In vivo, curzerene was found to significantly inhibit tumor growth in nude mice and to prolong the survival time of tumor‐bearing nude mice.ConclusionIn conclusion, inhibition of GSTA4 correlates with positive outcomes in glioma models, and thus, this molecule is a candidate drug for the treatment of glioma.     

In vivo gamma imaging of the secondary tumors of transplanted human fetal striatum neural stem cells-derived primary tumor cells

This study describes gamma-imaging of the secondary tumors from the transplanted human fetal striatum neural stem cells-derived primary tumor cells in nude mice. The subcutaneous primary tumors were detected to express integrin alphavbeta3, and the corresponding cells were isolated and enriched in vitro, then transplanted to the nude mice. The technetium-99m-labeled Arg-Gly-Asp peptide, with high affinity to integrin alphavbeta3, was prepared for biodistribution and gamma-imaging. The secondary tumors were readily visualized at 1-h postinjection, and the tumor uptake of radiotracer was similar to that of positive control animals transplanted with U87MG human glioma cells. The tumor specificity of radiotracer was demonstrated by blocking experiment. We concluded that gamma-imaging is a promising approach in imaging the tumorigenesis of transplanted stem cells in vivo.     

重组VEGF165-PE38融合基因对裸鼠移植性人胶质瘤血管生成的抑制作用

目的 研究血管内皮细胞生长因子(VEGF)和铜绿假单胞菌外毒素A(PE)融合基因的真核表达载体对裸鼠移植性人脑恶性胶质瘤血管生成的影响,探索抗肿瘤血管生成的新方法.方法 采用裸鼠背部皮下注射U251细胞建立移植性恶性胶质瘤模型,9 d后按随机数字表法将裸鼠分为未治疗组、PBS组、空质粒组、重组质粒组,采用HE染色观察肿瘤组织的形态学变化.免疫组织化学SP法检测胶质纤维酸性蛋白(GFAF)、CD31、PE的表达.分析肿瘤组织的微血管密度(MVD). 结果 注射后第16天重组质粒组裸鼠移植瘤体积明显小于其他3组,差异有统计学意义(P<0.05);重组质粒组裸鼠移植瘤MVD低于其他3组,差异均有统计学意义(P<0.05);重组质粒组裸鼠肿瘤组织PE呈阳性表达,而在空质粒组、PBS组及未治疗组均为阴性表达. 结论 VEGF165-PE38融合基因的表达产物对人脑恶性胶质瘤有明显的抑制作用,并能抑制肿瘤新生血管生成,可能为一种有效抗肿瘤血管治疗的新策略.     

反义miR-21抑制异种移植U87人脑胶质瘤生长的体内研究

目的 探讨敲低miR-21表达抑制裸鼠皮下荷U87人脑胶质瘤生长的疗效和机制.方法 原位注射miR-21反义寡聚核苷酸(AS-miR-21)治疗裸鼠皮下荷U87人脑胶质瘤.定时测量肿瘤大小评估原位注射AS-miR-21的治疗效果.使用RT-PCR和原位杂交方法 鉴定治疗后miR-21表达水平,采用HE染色和免疫组织化学染色(增殖细胞核抗原、细胞周期抑制因子-21、基质金属蛋白酶-9和隔蛋白-7)评价治疗后肿瘤生物学性状的变化,TUNEL法检测肿瘤细胞凋亡. 结果 肿瘤生长曲线显示AS-miR-21治疗组肿瘤生长速度及体积明显小于对照组与无义序列治疗组,差异有统计学意义(F=6.056,P=0.007);RT-PCR检测显示AS-miR-21治疗组miR-21表达下调为对照组的(0.031±0.008)%;原位杂交显示AS-miR-21治疗组miR-21表达水平较对照组与无义序列治疗组下调:组织病理学检测表明AS-miR-21治疗后肿瘤恶性度降低;TUNEL法检测可见AS-miR-21治疗组细胞凋亡数明显高于对照组与无义序列治疗组,差异有统计学意义(F=141.021,P=0.000). 结论 以miR-21作为靶点治疗异种移植U87人脑胶质瘤效果令人满意,miR-21可作为人脑胶质瘤基因治疗的侯选靶点.     

Inhibitory Effect of IFN-��, on the Antitumor Activity of Celecoxib in U87 Glioma Model

Objective

Interferon-β, (IFN-β) has been used in the treatment of cancers. Inhibition of the enzyme cyclooxygenase (COX) with celecoxib had a significantly suppressive effect on tumor growth, angiogenesis, and metastasis in a variety of tumors. The aim of this study was to elucidate the antiglioma effect of combined treatment with IFN-β and celecoxib in U87 glioma model.

Methods

The in vitro effects of IFN-β (50-1,000 IU/mL) and celecoxib (50-250 µM) alone or combination of both on the proliferation and apoptosis of U87 cells were tested using MTT assay, FACS analysis and DNA condensation. To determine the in vivo effect, nude mice bearing intracerebral U87 xenograft inoculation were treated with IFN-β intraperitoneally (2×10⁵ IU/day for 15 days), celecoxib orally (5, 10 mg/kg) or their combination.

Results

IFN-β or celecoxib showed an inhibitory effect on the proliferation of U87 cells. When U87 cells were treated with IFN-β and celecoxib combination, it seemed that IFN-β interrupted the antiproliferative and apoptotic activity of celecoxib. No additive effect was observed on the survival of the tumor bearing mice by the combination of IFN-β and celecoxib.

Conclusion

These results suggest that IFN-β seems to inhibit the antiglioma effect of celecoxib, therefore combination of IFN-β and celecoxib may be undesirable in the treatment of glioma.     

过表达MiR-494抑制脑胶质瘤细胞U87的增殖

目的观察微小RNA-494(miR-494)对脑胶质瘤细胞U87增殖能力的影响。方法通过实时荧光定量聚合酶链式反应(qRT-PCR)法检测5例瘤旁组织和14例胶质瘤样本中miR-494的表达水平,并且检测其在6种胶质瘤细胞系中的表达;将100 nmol/L miR-494 mimics瞬时转染至脑胶质瘤U87细胞,qRT-PCR检测miR-494表达情况;采用四唑盐比色法(MTT)检测U87细胞增殖情况;流式细胞术(FCM)检测对U87细胞周期的影响。结果与瘤旁脑组织相比,miR-494在胶质瘤中呈现低表达;U87细胞转染48 h后,miR-494 mimics组miR-494表达为对照组的357倍(P0.05);与对照组相比,miR-494 mimics转染后抑制了U87细胞的增殖能力(P0.05);S期细胞显著减少,而G1期细胞则明显增多。结论 miR-494在胶质瘤中低表达,过表达miR-494 mimics有效抑制了U87细胞的增殖,调节细胞周期S期降低G1期增多,提示miR-494有望成为胶质瘤治疗的分子靶点。     

SEPT7基因抑制U251 MG裸鼠皮下荷瘤侵袭的体内实验研究

目的 探讨SEPT7 基因在体内抑制胶质瘤生长和侵袭的作用.方法 建立人脑胶质母细胞瘤U251细胞来源的裸鼠皮下胶质瘤模型,并给予SEPT7治疗,定期测量肿瘤体积变化,检测肿瘤组织中SEPT7以及侵袭相关蛋白MMP2、MMP9、MT1-MMP、TIMP1、TIMP2和整合素α_v β_3的表达.结果 SEPT7 显著抑制肿瘤生长.治疗组肿瘤体积明显小于对照组(P＜0.01).SEPT7 治疗组中,肿瘤组织细胞内SEPT7表达明显上调.MMP2,MMP9,MT1-MMP和整合素α_v β_3的表达下降,而TIMP1、TIMP2 的表达明显升高.结论 SEPT7基因对于胶质瘤的侵袭和生长具有抑制作用.     

人脑胶质瘤浸润淋巴细胞抗肿瘤活性的实验和临床研究

目的检测人脑胶质瘤浸润淋巴细胞（ＧＩＬ）的来源及其抗肿瘤活性。方法把从人脑胶质瘤手术标本中提取的ＧＩＬ，在体外以白细胞介素２（ＩＬ２）刺激后培养扩增，将扩增后的ＧＩＬ注入人脑胶质瘤裸鼠动物模型体内观察其动物体内杀瘤活性。选择１１例术后再次复发的人脑胶质瘤晚期患者进行ＧＩＬ临床治疗。结果经ＩＬ－２刺激培养３周的ＧＩＬ杀瘤活性最强。裸鼠体内试验证实ＧＩＬ肿瘤局部注射能显著抑制肿瘤生长。临床治疗１１例患者随访２年仅１例复发。结论胶质瘤术后将ＧＩＬ培养扩增３周，再回输入肿瘤残腔杀灭残留肿瘤细胞，可能是一种较为理想的人脑胶质瘤综合疗法     

microRNA-328 is a favorable prognostic marker in human glioma via suppressing invasive and proliferative phenotypes of malignant cells

Objective: microRNA (miR)-328 has been reported to be implicated into tumorigenesis and tumor progression in human gliomas. However, there were controversial study results in relation to its expression pattern as well as functions in this disease. The aim of the current study was to determine the clinical significance of miR-328 expression in patients with gliomas and its effect in tumor cell malignant phenotypes. Methods: Quantitative real-time PCR was performed to detect the expression levels of miR-328 in 116 glioma and 15 non-neoplastic brain tissues. Then, the correlations of miR-328 expression with selected clinicopathologic parameters and clinical outcome of glioma patients were statistically evaluated. Moreover, CCK-8 and transwell assays were performed to investigate the functions of miR-328 in cell proliferation, invasion and migration, respectively. Results: Compared to non-neoplastic brain tissues, the expression levels of miR-328 were significantly downregulated in glioma tissues (p < 0.001). In addition, miR-328 downregulation was significantly associated with WHO grade (p < 0.001) and Karnofsky performance status score (p = 0.02). Moreover, glioma patients with low miR-328 expression exhibited markedly shorter overall survival than those with high expression (p < 0.001). Furthermore, functional assays in vitro system demonstrated that enforced expression of miR-328 could notably attenuate cell proliferation, invasion and migration of two glioma cell lines, including U251 and U87. Conclusions: Our data offer the convincing evidence that loss of miR-328 expression may stimulate advanced tumor progression and adverse outcome via promoting cellular proliferation and invasion. We propose a tumor suppressive role of miR-328 and its potential therapeutic value in human glioma.     

TIMP-2基因对人脑胶质瘤U87细胞周期与增殖的影响

目的探讨金属蛋白酶组织抑制因子(TIMP-2)对人脑胶质瘤细胞的抑制作用,主要是对细胞周期与细胞增殖的作用.方法用含TIMP-2基因的重组腺病毒载体,体外转染人胶质瘤细胞系U87,用Western blot检测目的蛋白的表达.通过细胞生长实验,流式细胞仪分析技术检测U87转染前后细胞增殖、周期与凋亡的变化.结果转染AdTIMP-2病毒后的U87细胞TIMP-2蛋白表达上调,转染后对U87细胞的生长有抑制作用,并出现明显的G0 -G1 期阻滞,但无明显的细胞凋亡峰出现.结论重组腺病毒载体介导的TIMP-2基因在体外对人脑胶质瘤细胞系U87 的生长有抑制作用,可作为胶质瘤治疗的有效工具.