绿色荧光蛋白表达对检测胶质细胞瘤体内侵袭的意义

利用体外转染绿色荧光蛋白基因的胶质瘤细胞大鼠移植瘤模型,研究胶质瘤体内侵袭行为。方法：携有增强型绿色荧光蛋白（ＥｎｈａｎｃｅｄＧｒｅｅｎＦｌｕｏｒｅｓｃｅｎｔＰｒｏｔｅｉｎ,ＥＧＦＰ）基因的ｐＥＧＦＰ－Ｎ３质粒体转染Ｃ６胶瘤细胞,筛选稳定表达绿色荧光蛋白的细胞克隆,并作流式细胞仪和电镜检测,以立体定向法植入ＳＤ大鼠脑实质内建立大鼠移植模型。４周后处死大鼠并作鼠脑连续石蜡切片,相邻切片分别作苏木素－     

人脑胶质细胞癌中血管内皮生长因子基因的表达研究

目的 研究血管内皮生长因子（ｖａｓｃｕｌａｒ ｅｎｄｏｔｈｅｌｉａｌ ｇｒｏｗｔｈ ｆａｃｔｏｒ,ＶＥＧＦ）在脑胶质细胞癌中的表达及其与肿瘤病理分级、新生血管形成的关系。方法 采用Ｎｏｒｔｈｅｎ杂交和免疫组织化学染色检测３０例脑胶质瘤标本,２例脑胶质瘤细胞株,４例正常脑组织的ＶＥＧＦ ｍＲＮＡ和蛋白表达水平。结果 正常脑组织中ＶＥＧＦ ｍＲＮＡ的表达水平极低,而脑胶质瘤组织和细胞系中ＶＥＧＦ ｍＲ     

重组人血管内皮抑素对食管癌细胞生长抑制作用的研究

目的　探讨重组人血管内皮抑素（恩度）对食管癌细胞生长的抑制作用。方法　用ＭＴＴ法检测恩度对食管癌细胞-１０９和人脐静脉内皮细胞（ＨＵＶＥＣ）的增殖抑制作用;建立Ｅｃａ-１０９细胞裸鼠移植瘤模型,观察不同浓度恩度对移植瘤生长的影响;免疫组织化学检测瘤组织微血管密度（ＭＶＤ）;ＥＬＩＳＡ方法检测荷瘤动物血清ＶＥＧＦ的浓度。结果　与空白对照组比较,恩度能抑制Ｅｃａ-１０９和ＨＵＶＥＣ细胞的增殖（Ｐ均＜０.０5）,并有效抑制食管癌移植瘤的生长（Ｐ＜０.０５）,检测荷瘤组织中ＭＶＤ计数较空白对照组明显降低（Ｐ＜０.０５）,荷瘤动物血清ＶＥＧＦ浓度也明显低于空白对照组（Ｐ＜０.０５）。结论 恩度能有效抑制食管癌细胞及移植瘤的生长。     

C-myc抑制肺癌细胞的肿瘤发生并且下调血管内皮生长因子表达[英]

Ｃｍｙｃ癌基因在源自肺肿瘤的细胞中频繁地扩增并且同这些癌症的恶性有关。为支持这一点,Ｃｍｙｃ转染提高人类小细胞肺癌(ＳＣＬＣ)细胞体外增殖与软琼脂中的克隆形成。在该研究中,作者惊讶地发现Ｃｍｙｃ表达抑制了ＳＣＬＣ细胞在无胸腺鼠中的肿瘤形成。Ｃｍｙｃ表达下调了这些ＳＣＬＣ细胞内血管内皮生长因子(ＶＥＧＦ)的蛋白表达与转录,以及Ｃｍｙｃ表达控制的鼠成纤维细胞和Ｃｍｙｃ转基因鼠肝细胞内的ＶＥＧＦ的转录。最后,双变量和多变量分析证实肺癌细胞系瘤形成可能性与Ｃｍｙｃ的相对表达呈负相关,与ＶＥＧＦ的相对表达呈正相关,…     

VEGF及其受体flt对荷瘤鼠喉癌细胞增殖的影响

目的;观察血管内皮生长因子（ＶＥＧＦ）对喉癌细胞增殖动力学的影响。方法：首先建立荷瘤（ＨＥＰ－２喉癌细胞）裸鼠动物模型,将抗ＶＥＧＦ抗体及抗ＶＥＧＦ受体ｆｌｔ抗体分别注射荷瘤鼠体内,通过活栓标记ＢｒｄＵ的方法,观察抗ＶＥＧＦ抗体及ｆｌｔ抗体对喉癌ＨＥＰ－２细胞增殖的影响。结果：与对照组相比较,抗ＶＥＧＦ抗体治疗组ＨＥＰ－２细胞ｂｒｄＵ标记阳性率显著降低;     

血管内皮生长因子与抗血管治疗胶质瘤

血管内皮生长因子（ｖａｓｃｕｌａｒｅｎｄｏｔｈｅｌｉａｌｇｒｏｗｔｈｆａｃｔｏｒ，ＶＥＧＦ）具有增加血管通透性，促进血管内皮细胞增殖分裂的作用。研究证实〔１，２〕ＶＥＧＦ是肿瘤血管形成的主要促进因子，血管形成是肿瘤生长的关键，对实体瘤的快速生长和转...     

血管内皮生长因子抗体对实验性肿瘤的抑制作用

目的选择在肿瘤血管形成过程中较为重要的因子即血管内皮生长因子（ｖａｓｃｕｌａｒｅｎ－ｄｏｔｈｅｌｉａｌｇｒｏｗｔｈｆａｃｔｏｒ，ＶＥＧＦ）为靶，进行抗肿瘤形成的实验性研究。方法应用亲和层析纯化的有中和活性的ＶＥＧＦ抗体进行实验性肿瘤的抑制研究。结果抗ＶＥＧＦ抗体对小鼠Ｓ１８０肉瘤的抑制作用呈明显的剂量依赖关系，当剂量为每只２００μｇ／ｄ时，抑瘤率为４１．０％，在裸鼠移植人胃癌（ＭＧＣ８０３）模型中，ＶＧＥＦ抗体的抑瘤率可达７６．２％，如合并使用胃癌单克隆抗体与１３１Ｉ偶合物（１３１Ｉ－３Ｈ１１），可使４／５的裸鼠无瘤生存，其抑瘤率较单独使用偶合物增加８４．０％。结论ＶＥＧＦ抗体对肿瘤治疗具有潜在的应用价值。     

抗VEGF抗体及抗flt抗体对人喉癌HEP-2细胞系的生长抑制作用

目的：观察抗ＶＥＧＦ抗体及抗ｆｌｔ抗体对ＨＥＰ－２人喉癌细胞生长的影响,探讨ＶＥＧＦ对ＨＥＰ－２细胞的作用及作用方式。方法：将不同浓度的抗ＶＥＧＦ及抗ｆｌｔ抗体分别加入ＨＥＰ－２细胞的培养液中,培养５天后测ＨＥＰ－２细胞的活性（ＭＴＴ法）。结果：不同浓度的ＶＥＧＦ抗体作用于ＨＥＰ－２细胞时,浓度从１０μｇ／ｍｌ至１μｇ／ｍｌ时,对ＨＥＰ－２细胞的生长具有显著的抑制作用（Ｐ〈０．０１）,当ＶＥＧＦ浓     

表皮生长因子受体反义cDNA对C6鼠脑胶质瘤体内治疗的研究

目的研究表皮生长因子受体（ＥＧＦＲ）反义ｃＤＮＡ对Ｃ６鼠脑胶质瘤的体内治疗效果。方法将野生型及已转染ＥＧＦＲ反义ｃＤＮＡ的Ｃ６鼠胶质瘤细胞接种于鼠脑右侧尾状核（对照组８只,转染组６只）,并对鼠脑内已形成的Ｃ６胶质瘤用脂质体包裹的ＥＧＦＲ反义ｃＤＮＡ瘤区原位注射（治疗组９只）。观察各组大鼠的一般情况、生存期、肿瘤病理学和磁共振成像（ＭＲＩ）动态改变,采用Ａｇ－ＮＯＲ计数、ＴＵＮＥＬ法检测肿瘤细胞增殖活性及凋亡。结果对照组８只大鼠平均生存期为１７．３天,转染组６只及治疗组６只大鼠生存期明显延长,除因病理检查人为处死外,无自然死亡,存活期超过２００天。ＭＲＩ检查对照组大鼠脑内有明显瘤灶,转染组大鼠未形成瘤灶,治疗组大鼠脑内瘤灶治疗后消失,均与病理学检查结果一致。而且治疗组大鼠Ｃ６细胞增殖活性降低,大量细胞凋亡,ＥＧＦＲ表达减少。结论ＥＧＦＲ可望成为基因治疗的优选靶基因     

自杀基因治疗恶性胶质瘤的研究

目的：单纯疱疹病毒Ⅰ型胸苷激酶（ＨＳＶ－ｔｋ）基因治疗恶性胶质瘤体内外试验。方法：分子克隆及真核细胞基因转染技术构建逆转录病毒（ＲＶ）载体ｐＭＶ７（ｔｋ）及ＰＡ３１７ｔｋ包装细胞系;体外不同比例混合鼠Ｃ６胶质瘤细胞与ＰＡ３１７ｔｋ细胞,在ＧＣＶ（Ｇａｎｃｉｃｌｏｖｉｒ）作用下观察细胞存活率;建立ＳＤ大鼠颅内Ｃ６胶质瘤模型（种植５×１０５Ｃ６细胞）,治疗组第３天原位注射５×１０６ＰＡ３１７ｔｋ细胞,５天后腹腔给予ＧＣＶ（３０ｍｇ／ｋｇ．ｄ）,ＭＲＩ全程监测肿瘤消长,观察病症及存活期,并行病理检查。结果：在ＧＣＶ０．１～１０１μｇ／ｍｌ浓度范围内,Ｃ６细胞存活率随ＰＡ３１７ｔｋ细胞混入比例增加而逐渐减低（Ｐ＜０．００１）,并具有ＧＣＶ剂量依赖性（Ｐ＜０．０１）;体内试验治疗组病症轻,生存期延长,ＭＲＩ表明治疗组肿瘤体积较对照组明显减小（Ｐ＜０．０１）,１个月时病理检查见肿瘤细胞消失,代之以小胶质细胞增生并形成坏死囊。结论：应用本实验室构建的ＲＶ载体ｐＭＶ７（ｔｋ）及其ＰＡ３１７ｔｋ包装细胞系治疗恶性胶质瘤是一种有效及具有前途的治疗方法。     

血管内皮生长因子抗体与实验性肿瘤的抑制作用

选择在肿瘤血管形成过程中较为重要的因子血管内皮生长因子（ｖａｓｃｕｌａｒｅｎｄｏｔｈｅｌｉａｌｇｒｏｗｔｈｆａｃｔｏｒ,ＶＥＧＦ）为靶,进行抗肿瘤形成的实验研究。方法应用亲和层析纯化的有中和活性的ＶＥＧＦ抗体进行实验性肿瘤的抑制研究。结论ＶＥＧＦ抗体对肿瘤具有潜在的应用价值。     

小鼠sFlt重组腺病毒抑制小鼠Lewis肺癌的生长

背景与目的实体肿瘤组织的生长具有血管依赖性，肿瘤新生血管生成已证实为肿瘤生长的控制点之一。本研究拟探讨编码小鼠可溶性血管内皮生长因子受体1（sFlt1）的复制缺陷型腺病毒对肿瘤新生血管和肿瘤生长的影响。方法应用小鼠Lewis肺癌细胞（LLC）株建立肺癌模型进行抗肿瘤的研究，以编码sFlt1的复制缺陷型重组腺病毒（sFlt1-Adv）作为治疗组，编码绿色荧光蛋白的复制缺陷型重组腺病毒（GFP—Adv）和生理盐水作为对照组，皮下接种小鼠LLC一周后，经尾静脉给药2次（间隔2周），第二次给药后2周，处死全部实验鼠，剥离肿瘤组织并称重，用3％多聚甲醛固定，用抗CD31作免疫组织化学染色。结果sFlt1—Adv治疗组肿瘤明显小于GFP—Adv对照组和生理盐水组（P％0．01），其抑瘤率达到71．8％；治疗组的肿瘤微血管密度低于GFP-Adv对照组和生理盐水组（P％0．01）。结论复制缺陷型重组腺病毒能抑制肿瘤组织的新生血管形成，从而抑制肿瘤的牛长，有较好的应用价值。     

血管生成抑制素对C6脑胶质瘤的抑瘤效应

目的探讨不同剂量的血管生成抑制素（Angiostatin）在胶质瘤治疗中的抑制血管生成作用。方法应用不同剂量（100ng、1000ng）的血管生成抑制素分别作用于体外培养的大鼠C6脑胶质瘤细胞系和C6/SD大鼠脑胶质瘤模型,分别计算细胞存活率和抑瘤生成率,SABC免疫组织化学技术检测体内胶质瘤中的微血管密度。结果不同剂量的血管生成抑制素均对体外培养的胶质瘤细胞的生长不产生明显抑制作用;对体内生长的胶质瘤则有显著抑制作用,而且较高剂量的Angiostatin的抑制作用更为明显,抑瘤率最高达65.3％（P<0.01）。经不同剂量的血管生成抑制素处理的脑胶质瘤中微血管密度较对照组降低（P<0.01）。结论血管生成抑制素能抑制C6脑胶质瘤的血管生成,且较高剂量实验组的作用更为明显,此研究对血管生成抑制素抑制C6胶质瘤的生长机制研究奠定了一定基础。     

Anti-Vascular endothelial growth factor treatment augments tumor radiation response under normoxic or hypoxic conditions

Recent studies in experimental animals have shown that combining antiangiogenic therapy with radiation can enhance tumor response. Whether this enhancement is mainly attributable to angiogenesis inhibition, endothelial cell radiosensitivity, tumor cell apoptosis, or a decrease in the number of hypoxic cells (improved oxygenation) is not known. We designed this study to discern the role of tumor oxygenation. We chose an anti-vascular endothelial growth factor (anti-VEGF) monoclonal antibody (mAb) which has a known target, human VEGF. We also measured interstitial fluid pressure (IFP) to test the hypothesis that the decreased vascular permeability induced by the anti-VEGF mAb can lower IFP. The effect of anti-VEGF mAb on vascular density, partial oxygen tension (pO2), and apoptosis was also measured. Athymic NCr/Sed nu/nu mice bearing 6-mm xenograft of the human glioblastoma multiforme (U87), or colon adenocarcinoma (LS174T) were treated with anti-VEGF mAb injected i.p. on alternate days for a total of six injections at a dosage of 100 microg/injection/mouse. For combined anti-VEGF and radiation, single radiation doses were given under normal blood flow (20 and 30 Gy) or clamped hypoxic conditions (30 and 40 Gy) 24 h after the sixth injection of mAb. The inhibition of the growth of U87 and LS174T tumors by the anti-VEGF mAb was associated with a significant reduction in tumor vascular density and a relatively small increase in the number of apoptotic cells. Compared with size-matched controls, IFP decreased by 74% in LS174T, and 73% in U87 in mice treated with anti-VEGF mAb. After antibody treatment PO2 increased significantly in U87, but did not change in LS174T tumors. Combined treatment induced in U87 tumors a tumor-growth delay (TGD) which was greater than additive; in LS174T except for the 40-Gy hypoxic group, the effect was only additive. In both U87 and LS174T the TGD induced by the antibody was independent of oxygen levels in the tumor at the time of radiation. The fact that the increase in TGD occurred under both normoxic and hypoxic conditions suggests that anti-VEGF mAb treatment can compensate for the resistance to radiation induced by hypoxia.     

表达抗-VEGF发夹状核酶腺病毒载体的构建及其对结肠癌生长的抑制作用

背景与目的：VEGF过表达提示结肠癌预后不良。本实验构建带有抗-VEGF发夹状核酶的复制缺陷型腺病毒，观察其对结肠癌VEGF表达及肿瘤生长的抑制作用。方法：将人工合成的抗-VEGF发夹状核酶DNA定向克隆至转移质粒pAdTrack—CMV多克隆位点，然后与病毒骨架质粒pAdEasy-1在细菌BJ5183中重组，在293a细胞中包装成完整病毒（命名为Ad-Rz）并复制扩增、纯化。RT-PCR检测病毒感染后抗-VEGF发夹状核酶在HT-29细胞中的表达、real-time PCR及ELISA检测其对VEGF mRNA及蛋白表达的抑制作用。观察Ad-Rz对HT-29细胞和裸鼠移植瘤生长的抑制作用，CD34标记检测肿瘤组织微血管密度（MVD）。结果：抗-VEGF发夹状核酶成功克隆至复制缺陷型腺病毒载体上。抗-VEGF发夹状核酶可在HT-29细胞中表达．Ad—Rz感染的HT-29细胞中VEGFmRNA的相对表达量大约为PBS组的45％（e〈0．05）。ELISA结果显示Ad—Rz感染后的细胞上清液中蛋白含量（A值）为0．455±0．35／百万细胞，低于PBS组（P〈0．05）。Ad—Rz对HT-29细胞的生长抑制无统计学意义（P〉0．05），但可显著抑制肿瘤组织内血管的形成（P〈0．05），Ad—Rz治疗的移植瘤增殖率低于PBS组，但差异无统计学意义（P〉0．05）。结论：腺病毒载体介导的抗-VEGF发夹状核酶可有效抑制HT-29细胞VEGF的表达及移植瘤组织内血管的生成。     

Intratumoral neovascularization and growth pattern in early gastric carcinoma.

BACKGROUND: The growth pattern of early gastric carcinoma, based on a volumetric analysis, reflects biologic characteristics of the tumor. The authors investigated the microvessel density (MVD), expression of vascular endothelial growth factor (VEGF), and growth patterns in early gastric carcinoma. METHODS: Ninety-four tissue specimens resected from patients with early gastric carcinoma invading the submucosal layer were examined. Microvessel quantification was performed immunohistochemically using a monoclonal antibody against factor VIII-related antigen. VEGF expression was studied using an anti-VEGF polyclonal antibody. Growth patterns were defined as follows: Pen A type: expansively penetrating growth; Pen B type: infiltratively penetrating growth; Super type: superficially spreading growth. RESULTS: The mean MVD was 16.9 (range, 5.2-43.0). MVD was significantly higher in tumors with venous invasion (P<0.01), lymphatic vessel invasion (P<0.05), and lymph node metastases (P<0.05) compared with MVD in tumors without venous or lymphatic vessel invasion or lymph node metastases. The VEGF-positive rate of Pen A type tumors was 66.7% (18 of 27), that Pen B type was 10.0% (1 of 10), that of Super type was 19.4% (6 of 31), and that of the unclassified type was 15.4% (4 of 26). The VEGF-positive rate in patients with Pen A type tumors was significantly higher than that in patients with the other three growth patterns(P<0.01). MVD in patients with Pen A type tumors (25.9+/-9.2) was significantly higher than that in patients with Super type tumors (12.6+/-5.4) (P<0.01). Patients with Pen A type tumors had a poorer prognosis than patients whose tumors had other growth patterns (P<0.05). According to multivariate analysis, VEGF expression and lymphatic vessel invasion were significant prognostic factors. CONCLUSIONS: Pen A type gastric carcinoma tends to secrete VEGF, thus inducing tumor angiogenesis and resulting in venous invasion. Intensive follow-up is necessary for patients with Pen A type tumors, because this tumor type has a greater propensity for hematogenous metastasis.     

Growth Control of C6 glioma in vivo by Nerve Growth Factor

Treatment with nerve growth factor (NGF) causes differentiation of rat C6 glioma cells and strongly inhibits their proliferation in vitro. This suggests that induction of NGF-mediated differentiation may provide a novel therapeutic approach to growth control of glial tumors. We examined the effects of NGF treatment on the growth potential of C6 glioma, which expressed NGF receptor in vivo. C6 glioma cells (1 × 10⁶ cells/10 l) were transplanted into the rat striatum. After 4 days, the animals were given successive injections of 100 ng NGF into the growing tumor at every 4 days (n = 10 rats). Controls were subjected to identical procedures with vehicle which did not contain NGF (n = 10 rats). At 14 days after transplantation, we evaluated the tumor volume, proliferative cell index (PCI) based on the MIB-1 immunoreactivity and enzyme activities related to energy metabolism by enzyme histochemistry. We found that the NGF treatment markedly reduced the tumor volume of the C6 glioma (34.00 ± 8.47 mm³ to 7.22 ± 4.92 mm³, p < 0.01). NGF treatment also decreased the PCI (33.34 ± 9.57% to 3.85 ± 3.56%, p < 0.01) with a negative correlation with tumor volume (r = 0.972, p < 0.01), and the hexokinase (HK) and glucose-6-phosphate dehydrogenase (G6PDH) activities (p < 0.01 and p < 0.01, respectively) which reflect the demand for nucleic acid synthesis for proliferation through the glycolytic and pentose phosphate pathways. The present results demonstrate for the first time that inhibition of tumor cell proliferation of C6 glioma by NGF occurs in vivo, probably through the NGF-mediated differentiation of C6 glioma cells which has been observed in in vitro studies.     

Dual Pathways to Tubular Morphogenesis of Vascular Endothelial Cells by Human Glioma Cells: Vascular Endothelial Growth Factor/Basic Fibroblast Growth Factor and Interleukin-8

In this study, we examined whether human glioma cells are angiogenic in a model using human microvascular endothelial cells, and also which factor is responsible for the glioma-dependent angiogenesis. Tubular morphogenesis in type I collagen gel by human microvascular endothelial cells was stimulated in the presence of 10 and 100 ng/ml of vascular endothelial growth factor (VEGF), 10 ng/ml basic fihroblast growth factor (bFGF) and 10 ng/ml of interleukin-8 (IL-8). Tube formation of the microvascular endothelial cells was assayed in the glioma cell lines IN157 and IN301, co-cultured using the double chamber method. IN301 cells had much higher levels of VEGF, bFGF and transforming growth factor-(mRNA than IN157 cells, whereas the two had similar levels of transforming growth factor-Alfa mRNA. By contrast, IN157 cells had much higher levels of IL-8 mRNA than IN301 cells. IN301-dependent tubular morphogenesis was inhibited by anti-VEGF or anti-bFGF antibody, and the inhibition was almost complete when anti-VEGF and anti-bFGF antibodies were present. On the other hand, IN157-dependent tubular morphogenesis was inhibited by anti-IL-8 antibody, but not by anti-VEGF or anti-bFGF antibodies. These findings demonstrated dual paracrine controls of tumor angiogenesis by human glioma cells. One is mediated through VEGF and/or bFGF, and the other, through IL-8.     

Pharmacology and pharmacodynamics of bevacizumab as monotherapy or in combination with cytotoxic therapy in preclinical studies

Preclinical models have examined the pharmacologic and pharmacodynamic activities of an anti-vascular endothelial growth factor (VEGF) humanized, monoclonal antibody, bevacizumab, and/or its murine equivalent A4.6.1. These studies found that single-agent therapy with bevacizumab/A4.6.1 resulted in tumor growth inhibition of 20 different human tumor cell lines (13 tumor types) implanted into nude mice irrespective of the route of administration or tumor location. Several of these studies also observed significant inhibition of tumor metastases. Various studies have examined the feasibility of combining anti-VEGF therapy with cytotoxic or biological agents. Combining bevacizumab/A4.6.1 with doxorubicin, topotecan, paclitaxel, docetaxel, or radiotherapy resulted in additive or synergistic tumor growth inhibition. Changes in vascular functions were frequently reported, including decreased vessel diameter, density, and permeability in response to treatment. A reduction in interstitial fluid pressure was also observed. In some studies, these improvements resulted in an increase in intratumoral uptake of chemotherapy, implying that the most effective use of anti-VEGF therapy is in combination with chemotherapy. Alternatively, combination treatment with radiation increased tumor oxygenation and tumor growth inhibition. Interestingly, anti-VEGF therapy has also been reported to reduce the development of ascites in ovarian mouse models. Finally, safety pharmacology studies with bevacizumab in cynomolgus monkeys showed that this agent is generally well tolerated with no unexpected adverse events.