重组人分泌型endostatin的纯化及其抑癌活性探讨

目的：纯化毕赤酵母菌GSll5株表达的分泌型人endostatin蛋白，并对其抑癌活性进行研究。方法：利用SepharoseG—25及Heparin亲和层析柱纯化重组蛋白，并以MTT法测定重组endostatin对人脐静脉血管内皮细胞(ECV-304)增殖的抑制活性；另以HepG2.2．15细胞注射裸鼠成瘤后，体内验证重组蛋白的抑瘤活性。结果：MTT结果显示纯化后的endostatin在体外可特异性抑制人脐静脉内皮细胞的增殖，最高抑制率为86．0％；动物实验证明其可明显抑制荷瘤动物肿瘤的生长。结论：经纯化后的重组endostatin具有较强抑癌活性，将为临床抗血管生成治疗实体瘤研究奠定基础。     

瘤内注射pSTbAT-脂质体复合物对裸鼠移植瘤抑制效应的实验研究

目的：构建血管生成抑制因子arresten基因的真核表达载体，并观察瘤内注射质粒DNA-脂质体复合物对裸鼠移植瘤生长的影响。方法：利用PCR方法，由重组质粒pGEM-Arr中扩增出基因；将该基因定向克隆于真核表达载体中。20只裸鼠皮下注射Hepp2细胞，成瘤后以成功构建的质粒DNA-脂质体复合物瘤内注射，治疗后采用免疫组化方法检测肿瘤血管密度，并利用arresten基因瘤内注射的方法对抑制肿瘤的效果进行分析。结果：我们成功构建arresten基因真核表达载体(psTbAT)。实验组肿瘤生长速度较对照组慢(实验组平均15．3个阳性血管／10个视野，对照组平均112．5个阳性血管／10个视野)。结论：瘤内注射arresten基因质粒-DNA复合物能抑制裸鼠移植瘤的血管生成，并能抑制移植瘤的生长。     

肝素和氢化可的松对人肺癌抑瘤作用的体内实验研究

目的：研究肝素和氢化可的松的联合应用对人肺癌裸鼠皮下移植瘤生长抑制作用和对肿瘤血管生成、肿瘤细胞增殖及凋亡的影响，探讨肝素和氢化可的松联合应用抑制肿瘤血管生成的可能性。方法：建立人肺癌裸鼠皮下移植瘤模型，通过观察瘤体生长情况、免疫组化法测定微血管密度计数和肿瘤组织的增殖情况、电镜观察肿瘤组织凋亡情况和原位细胞凋亡检测，研究肝素和氢化可的松联合应用的抗血管生成和抑瘤作用。结果：肝素和氢化可的松治疗组瘤体生长较对照组明显受抑，且肿瘤组织微血管生成减少、增殖降低、凋亡增多。结论：肝素和氢化可的松联合应用具有明显的抗血管生成和抑瘤作用。     

泰素帝对荷瘤小鼠肿瘤组织VEGF和TSP1表达的影响

目的：观察泰素帝对裸鼠人大肠癌LOVO细胞皮下移植瘤血管生成抑制作用及其作用机理。方法：通过建立裸鼠人大肠癌LOVO细胞皮下移植瘤模型,观察泰素帝的抑瘤作用,并采用免疫组织化学法检测泰素帝对肿瘤组织MVD、VEGF、TSP1表达的影响。结果：泰素帝的抑瘤率为72．4％。泰素帝能显著降低肿瘤微血管密度,下调VEGF和上调TSP1的表达。结论：泰素帝可能通过抑制肿瘤血管生成而发挥抗肿瘤作用;下调VEGF可能是泰素帝抗血管生成作用机理之一;上调TSP1可能是泰素帝抗血管生成另一作用机理。     

抗血管内皮生长因子抗体抑制成骨肉瘤生长及血管生成的实验探讨

目的：观察抗血管内皮生长因子（VEGF）抗体对人成骨肉瘤生长的影响。方法：用抗VEGF单克隆抗体100μg隔天于治疗组荷瘤裸鼠肿瘤周围及瘤块内注射，对照组用等量PBS注射，共给药6次。测量两组裸鼠肿瘤体积及肿瘤湿重，肿瘤组织行免疫组织化学染色观察微血管密度（MVD），光镜下病理学检查。结果：治疗组与对照组的肿瘤体积、重量、MVD值两组间差异有显著性，抑瘤率达54．05％。病检发现治疗组肿瘤内有散在点状或线状坏死灶，甚至小片状坏死灶，对照组很少出现坏死灶。结论：抗VEGF单克隆抗体能够有效抑制裸鼠体内人成骨肉瘤移植瘤的血管生成抑制肿瘤生长．     

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。     

CH50多肽真核表达载体pCH510的构建、表达及体内趋化和抑瘤作用

目的：探讨血管内皮生长因子的表达与肿瘤血管生成的关系。方法：将VEGF165正、反义RNA表达载体导入人胃癌细胞,观察接种VEGF高表达和低表达胃癌细胞裸鼠移植瘤的生长情况,并对移植瘤进行组织学检查,检测其血管密度、组织增生及环死程度等变化。结果：VEGF正义转染细胞所致移植瘤的生长速度明显快于反义转染细胞所致的移植瘤;组织学检查发现,正义转染细胞移植瘤的血管密度显著高于的转染细胞所致的肿瘤。结论：血管皮生长因子通过启动血管生成而促进肿瘤的生长,阻断血管内皮生长因子的产生可以抑制肿瘤的生长。     

优福定节拍性化疗抗MKN-45荷瘤裸鼠血管生成作用的研究

目的：研究持续小剂量优福定（UFT）对MKN-45荷瘤裸鼠的疗效及其机制。方法：建立荷瘤动物模型，用UFT和环磷酰胺（CTX）对荷瘤动物模型进行治疗，观察其疗效及毒副作用；流式细胞术检测外周血内皮祖细胞（CEP）；免疫组化检测微血管密度（MVD）。结果：持续小剂量UFT明显抑制肿瘤的生长，CEP和MVD含量下降显著，而持续小剂量CTX虽然抑瘤率较持续小剂量UFT高，但却未抑制CEP和MVD含量。结论：持续小剂量UFT可通过抑制血管生成对MKN-45胃癌细胞荷瘤裸鼠产生抗瘤效应。     

EGCG对胃癌血管新生中VEGF信号通路的多靶点作用

目的：探讨EGCG对胃癌血管生成抑制作用及其信号通路。方法：建立裸鼠异位胃癌模型,经腹腔注射EGCG,检测肿瘤生长及肿瘤组织微血管密度;不同浓度EGCG处理胃癌细胞24 h,检测胃癌VEGF蛋白和mRNA表达及VEGF分泌;同时不同浓度EGCG处理脐静脉内皮细胞,检测内皮细胞生长、迁移和体外小管形成。结果：EGCG显著抑制胃癌生长和肿瘤血管生成,平均肿瘤抑制率60.4%;EGCG显著抑制胃癌VEGF蛋白、mRNA表达和VEGF分泌;EGCG时间和剂量依赖性地抑制VEGF诱导的内皮细胞增殖,同时也剂量依赖性地抑制VEGF诱导的内皮细胞的迁移和小管生成。结论：EGCG多靶点作用于VEGF信号通路,抑制胃癌生长和血管生成。     

纳米金阻断VEGF165信号传导并抑制裸鼠肝癌血管生成

目的:探讨纳米金如何阻断VEGF165的信号传导,抑制裸鼠移植肝癌血管生成.方法:无血清培养人脐静脉血管内皮细胞(HUVEC),加入血管内皮生长因子165(VEGF165),再加入不同浓度纳米金,作用5min,采用Western blot方法测定磷酸化PLC-γ1蛋白.6周龄Balb/c裸鼠20只,从裸鼠右腋皮下注入H22细胞.第5天,肿瘤形成约8mm大小,随机分为两组:实验组从肿瘤周围及瘤内注入纳米金,每天1次,连续8天;对照组用生理盐水处理.处死裸鼠时测量肿瘤体积及重量,免疫组化染色并计算肿瘤组织微血管密度.结果:VEGF165浓度不变(10μg/L),随着纳米金溶液浓度的增加(125、250、500nmol/L),纳米金抑制PLC-γ1磷酸化越来越明显.肝癌组织内微血管密度分别为,实验组14.27±1.08,对照组23.52±1.36,说明纳米金明显抑制了肝癌血管生成(P＜0.01).结论:纳米金阻断VEGF165的信号传导,明显抑制裸鼠肝癌血管生成.     

Endostatin 影响大肠癌裸鼠血管内皮生长因子及其受体机理的实验研究

目的探讨 Endostatin 对裸鼠大肠癌血管内皮生成因子(VEGF)及其受体(Flk-1)的抑制作用的机理.方法建立大肠癌裸鼠模型,将裸鼠分为2组,分别给予 Endostatin 和生理盐水治疗,15 d后处死裸鼠,采集肿瘤标本, 免疫组化法检测瘤株的 VEGF 和 Flk-1 的表达情况.结果Endostatin 对 VEGF 没有抑制作用(P＞0.05),对 Flk-1 有抑制作用(P＜0.05).结论Endostatin 通过竞争性抑制 VEGF 与其 Flk-1 结合实现抑制肿瘤生长的目的.     

毕赤酵母表达重组人内皮抑素对小鼠肺腺癌LA795生长和转移的抑制作用

背景与目的：肿瘤的生长和转移有赖于新生血管的生成,内皮抑素能抑制肿瘤的血管生成。本研究旨在观察毕巴斯德酵母（pichia.pastoris.GS115)分泌表达的重组人内皮抑素（recombinant human endostatin,rhES)对小鼠肺腺癌LA795生长和转换的抑制作用。方法：挑取一株高效分泌表达rhES的毕赤巴斯德酵母菌株,利用甲醇进行大量诱导表达;用肝素亲和层析的方法纯化目的蛋白;将接种LA795肺腺癌细胞的T739小鼠随机分成两组,分别给予rhES和PBS皮下注射,每日1次,连续14天;观察两组小鼠肿瘤生长情况,测量肿瘤体积大小,并观察两组小鼠肿瘤肺部转移情况。结果：经甲醇诱导的毕赤巴斯德酵母菌株高效分泌表达了rhES;动物实验研究发现rhES能显著抑制小鼠肺腺癌LA795的生长,rhES治疗组肿瘤大小与对照组比较具有显著性差异（P＜0．001）,抑瘤率达到66．4％,并且有效抑制了肿瘤的肺部转移。结论：利用毕赤酵母作为宿主分泌表达的rhES具有良好的生物学活性,可显著抑制小鼠肺腺癌LA795的生长和转移。     

血管内皮抑制素对Calu-6裸鼠移植瘤的生物学效应

目的 探讨血管内皮抑制素对Calu-6裸鼠移植瘤生长及新生血管形成的影响.方法 在荷瘤裸鼠皮下注射不同剂量的血管内皮抑制素,观察注射后肿瘤体积的变化;应用免疫组化SABC法检测肿瘤组织中血管内皮生长因子(VEGF)、生存素(survivin)和环氧化酶-2(COX-2)蛋白的表达以及微血管密度(MVD)的变化;流式细胞术检测循环血管内皮细胞(CECs)的含量;应用逆转录聚合酶链反应(RT-PCR)和实时定量PCR检测外周血中CD146和CD105 mRNA的表达.结果经血管内皮抑制素治疗后,荷瘤鼠肿瘤体积明显减小;肿瘤组织中VEGF、survivin和COX-2蛋白的表达以及MVD均下降,且各治疗组与阳性对照组间的差异均有统计学意义(均P＜0.05);外周血中CECs、CD146和CD105 mRNA的含量均明显下降;活化CECs的含量与肿瘤组织中survivin和VEGF的表达以及MVD的变化均呈正相关.结论 血管内皮抑制素可通过下调移植瘤中VEGF、survivin和COX.2蛋白的表达以及减少MVD抑制肿瘤生长;活化CECs将可能作为理想的预测抗血管形成治疗预后的标记物应用于临床.     

内皮抑素在直肠癌中的表达与血管生成的关系

目的:探讨内皮抑素(Endostatin)在直肠癌中的表达及其与血管生成的关系。方法:利用免疫组化S-P法,对40例直肠癌组织中的Endostatin表达进行研究。结果:Endostatin定位于癌组织中血管内皮细胞胞浆及胞膜,部分癌细胞胞浆也存在阳性表达,直肠癌组织中的Endostatin表达阳性率与正常直肠组织无明显差异(P>0.05),Endostatin表达与直肠癌临床病理中的分化程度、浸润深度无关(P>0.05),而与淋巴转移、Dukes分期相关(P<0.05)。结论:Endostatin在直肠癌的血管生成调控中具有重要作用。     

Endostatin overexpression inhibits lymphangiogenesis and lymph node metastasis in mice

Endostatin, a proteolytic fragment of collagen XVIII, is a potent inhibitor of angiogenesis and tumor growth. We studied the development of carcinogen-induced skin tumors in transgenic J4 mice overexpressing endostatin in their keratinocytes. Unexpectedly, we did not observe any differences in tumor incidence and multiplicity between these and control mice, nor in the rate of conversion of benign papillomas to malignant squamous cell carcinomas (SCC). We did find, however, that endostatin regulates the terminal differentiation of keratinocytes because the SCCs in the J4 mice were less aggressive and more often well differentiated than those in the control mice. We observed an inhibition of tumor angiogenesis by endostatin at an early stage in skin tumor development, but more strikingly, there was a significant reduction in lymphatic vessels in the papillomas and SCCs in association with elevated endostatin levels and also a significant inhibition of lymph node metastasis in the J4 mice. We showed that tumor-infiltrating mast cells strongly expressed vascular endothelial growth factor-C (VEGF-C), and that the accumulation of these cells was markedly decreased in the tumors of the J4 mice. Moreover, endostatin inhibited the adhesion and migration of murine MC/9 mast cells on fibronectin in vitro. Our data suggest that endostatin can inhibit tumor lymphangiogenesis by decreasing the VEGF-C levels in the tumors, apparently via inhibition of mast cell migration and adhesion, and support the view that the biological effects of endostatin are not restricted to endothelial cells because endostatin also regulates tumor-associated inflammation and differentiation, and the phenotype of epithelial tumors.     

内皮抑素对小细胞肺癌移植瘤微血管生成的影响

[目的]探讨内皮抑素对小细胞肺癌裸鼠移植瘤微血管生成的影响。[方法]将人小细胞肺癌细胞株NCI—H446接种于裸鼠背部皮下．待移植肿瘤成形后．随机分为对照组、试验1组和试验2组,每日于肿瘤部位分别注射生理盐水200μl、内皮抑素20μg、内皮抑素40μg,观察三组小鼠移植瘤的生长情况．并检测各组肿瘤的微血管密度。[结果]不同剂量的内皮抑素对肿瘤生长的影响均呈现明显的时间-效应和剂量-效应关系：随着内皮抑素剂量升高．移植瘤中的微血管密度下降。[结论]内皮抑素通过阻断微血管生成而抑制小细胞肺癌移植肿瘤的生长．     

Gene transfer of endostatin enhances the efficacy of doxorubicin to suppress human hepatocellular carcinomas in mice

Hepatocellular carcinoma (HCC) is one of the most common cancer-related causes of death, and is chemoresistant to anticancer drugs. Anti-angiogenic therapy has been shown to enhance the efficacy of chemotherapy to treat solid tumors. The aim of the present study was to determine whether endostatin, a potent antiangiogenic agent, could enhance the efficacy of doxorubicin to combat HCC. An endostatin expression plasmid was constructed and its expression in vitro and in vivo was detected after gene transfer. Recombinant endostatin inhibited angiogenesis in the chorioallantoic membrane assay, and showed synergistic effects with doxorubicin in inhibiting the in vitro proliferation of endothelial cells, but not that of tumor cells. Both endostatin gene therapy and doxorubicin suppressed the growth of subcutaneous human HepG2 tumors established in BALB/c nude mice, and tumor angiogenesis. Combination therapy with endostatin gene therapy and doxorubicin showed a stronger effect in suppressing tumor growth, and tumor angiogenesis, than the respective monotherapies. Gene transfer of endostatin down-regulated the expression of both hypoxia-inducible factor-1alpha and vascular endothelial growth factor (VEGF), whereas doxorubicin only down-regulated VEGF expression. Endostatin and doxorubicin synergized to down-regulate VEGF expression. Endostatin and doxorubicin combination therapy warrants investigation as a therapeutic strategy to combat HCC.     

Evaluation of endostatin antiangiogenesis gene therapy in vitro and in vivo.

Progressive growth and metastasis of solid tumors require angiogenesis, or the formation of new blood vessels. Endostatin is a 20-kDa carboxy-terminal fragment of collagen XVIII that has been shown to inhibit endothelial cell proliferation and tumor angiogenesis. Replication-deficient recombinant adenovirus (rAd) vectors were constructed, which encoded secreted forms of human and mouse endostatin (HECB and MECB, respectively), and, as a control, human alkaline phosphatase (APCB). Accumulation of endostatin was demonstrated in supernatants of cultured cells infected with the endostatin rAds. These supernatants disrupted tubule formation, inhibited migration and proliferation, and induced apoptosis in human dermal vascular endothelial cells or human vascular endothelial cells. Endostatin-containing supernatants had no effect on the proliferation of MidT2-1 mouse mammary tumor cells in vitro. A pharmacokinetic study of MECB in immunocompetent FVB mice demonstrated a 10-fold increase of serum endostatin concentrations 3 days after intravenous administration of 1x10(10) particles of this rAd (215-257 ng/mL compared to 12-38 ng/mL in control rAd-treated mice). Intravenous administration of MECB reduced b-FGF stimulated angiogenesis into Matrigel plugs by 38%. Intratumoral MECB inhibited growth of MidT2-1 syngeneic mammary tumors in FVB mice, but had minimal impact on the growth of MDA-MB-231 human breast tumors in SCID mice. Intravenous therapy with MECB also initially inhibited growth of MidT2-1 tumors, but this activity was subsequently blocked by induced anti-rAd antibodies. In summary, endostatin gene therapy effectively suppressed angiogenic processes in vitro and in vivo in several model systems.     

血管内皮抑素在非小细胞肺癌治疗中的研究进展

肿瘤血管生成及其生长、侵袭和转移是一个复杂的多因素多步骤的调控过程。肿瘤生长及转移依赖于新生血管形成，抑制血管形成是抗肿瘤治疗的新方法。血管内皮抑素是一种新型内源性血管生成抑制剂，体内外试验证明其能特异性抑制血管内皮细胞的增殖与迁移，诱导其凋亡，从而抑制肿瘤新生血管的形成，达到抑制肿瘤生长的目的。有关血管内皮抑素的具体作用机制及信号传导通路尚不十分明确。同时其表达水平与肿瘤具有明显的相关性．可作为肿瘤的临床预后指标。血管内皮抑素的基因治疗与化疗、放疗、免疫治疗联合应用不仅显著增强其他治疗抗肿瘤效应，而且可减少其他治疗的毒副反应，显示出良好的临床应用前景。其抗血管生成作用具有广谱、低毒的特点，更重要的是克服了肿瘤的耐药性，这对于人类最终克服肿瘤是非常重要的。多年来国内外对其用于非小细胞肺癌治疗的报道较多。本文就血管内皮抑素的结构、作用机制、在非小细胞肺癌中的临床前及临床试验研究做一综述。     

Localization of endostatin in rat and human gliomas

BACKGROUND: Endostatin is a potent inhibitor of endothelial cell proliferation, angiogenesis, and tumor growth. Its occurrence and localization has not yet been examined in human brain tumors. The authors report the production of a monoclonal antibody and detection of endostatin in rat and human gliomas by immunohistochemistry. METHODS: The authors analyzed localization and tissue distribution of endostatin in 41 paraffin embedded glioma samples (18 glioblastoma multiforme, 7 WHO Grade III astrocytomas, 13 fibrillary, and 3 protoplasmic WHO Grade II astrocytomas) of human origin and 21 rat C6 gliomas by immunohistochemistry. Double labeling experiments confirmed the origin of endostatin-labeled cells. RESULTS: Endostatin immunoreactivity was detected in tumor cells, endothelial cells, macrophages, and lymphocytes of both rat and human gliomas. The percentage of cells labeled with the endostatin antibody was significantly lower (P = 0.0126) in the tumor parenchyma of human glioblastomas than in WHO Grade II astrocytomas. CONCLUSIONS: Endostatin was present in various cell types in rat and human gliomas in vivo. Lower levels in glioblastomas than in WHO Grade II astrocytomas might have reflected the shift of a probable regulatory balance between promoters and inhibitors of angiogenesis towards facilitation of neovascularization.