腺相关病毒介导的人内皮抑素基因转染对肝细胞性肝癌生长的抑制作用

目的　研究腺相关病毒介导的人内皮抑素基因转染对肝细胞性肝癌的抑制作用。方法　用含人内皮抑素的腺相关病毒(r AAV2 /EGFP- Endo)转染肝癌细胞Hep3B,通过流式细胞仪检测其转染率。MTT法检测转染细胞的培养液上清对人脐静脉内皮细胞ECV30 4增生的抑制作用。Hep3B细胞接种裸鼠建立移植瘤模型,分别瘤内注射r AAV2 /EGFP- Endo(2×10 1 0 v.g.)、r AAV2 /EGFP或生理盐水,3周后检测裸鼠血液中内皮抑素的表达及移植瘤的体积和微血管密度(MVD)。结果　流式细胞仪结果显示重组腺相关病毒体外对Hep3B细胞的转染率为6 2 .5 % ;转染内皮抑素基因的Hep3B细胞的培养液上清能显著抑制ECV30 4细胞的生长(P<0 .0 1)。移植瘤内注射r AAV2 /EGFP- Endo后,荷瘤裸鼠血清中内皮抑素浓度为(82 .6 4±7.5 4 ) μg/L,移植瘤的体积和微血管密度均明显低于对照组(P<0 .0 1)。结论　腺相关病毒介导的人内皮抑素基因转染能够有效抑制人肝细胞性肝癌的生长。     

罗勒多糖对肿瘤转移行为的影响

目的:研究腺病毒介导的鼠内皮抑素基因对胃癌的治疗作用.方法:利用病毒重组技术将内皮抑素克隆入增殖缺陷型腺病毒基因组中,观察体外转染表达后的生物学活性.通过建立荷人胃癌的裸鼠动物模型,分析基因转导后动物肿瘤细胞中内皮抑素的表达情况及对肿瘤的抑制作用.结果:构建了表达内皮抑素的重组腺病毒载体pCA13-mEndo;检测到内皮抑素在体外的mRNA水平和蛋白质水平均有表达;鸡胚绒毛尿囊膜实验表明其对血管生长起抑制作用;荷瘤裸鼠体内肿瘤生长抑制明显.结论:所构建的pCA13-mEndo重组腺病毒载体可有效表达具有生物学活性的内皮抑素,使得肿瘤内微血管生成减少,肿瘤细胞增殖减慢,为抗血管生成方法治疗实体瘤的临床应用奠定基础.     

增殖缺陷型腺病毒介导人内皮抑素基因抗乳腺癌的实验

 目的通过建立裸鼠乳腺癌肿瘤模型,在体内实验中进一步证实携带人内皮抑素基因的增殖缺陷型腺病毒Ad-hE的抗肿瘤作用。方法在BALB/c裸鼠皮下种植人乳腺癌细胞MCF-7,建立移植瘤模型。在瘤体内注射Ad-hE治疗,观察肿瘤生长,免疫组化检测肿瘤细胞内皮抑素的表达和计数肿瘤间质中微血管的数量。结果Ad-hE具有明显抑制肿瘤细胞生长的作用,瘤体内注射重组腺病毒后4周,Ad-hE治疗组肿瘤体积为(225.3±90.2)mm3,明显小于Ad-LacZ病毒对照组(794.9±189.8)mm3和空白对照组(890.7±102.5)mm3。免疫组化显示,乳腺癌细胞在感染腺病毒后能够有效表达内皮抑素,阳性细胞比率超过60%以上。Ad-hE治疗组瘤组织中血管数量(16.3±7.3)明显少于空白对照组(54.6±17.6)和Ad-LacZ病毒对照组(49.8±21.2)。结论增殖缺陷型腺病毒介导人内皮抑素的表达,具有明显抑制肿瘤细胞生长的作用。     

SA脂质体介导血管抑素和／或内皮抑素基因治疗Lewis肺癌小鼠的实验研究

 目的 观察SA脂质体介导血管抑素和／或内皮抑素基因对Lewis肺癌小鼠移植瘤生长、转移的抑制作用。方法 建立C57BL／6j小鼠肺癌模型，30只荷瘤鼠随机分空白对照组，SA脂质体对照组，血管抑素基因（pAng）治疗组，内皮抑素基因（pEnd）治疗组，血管抑素和内皮抑素基因联合治疗组，每组6只。以SA脂质体介导，将血管抑素和／或内皮抑素基因直接注入移植瘤内，每周2次，共6周，每周测瘤体大小2次，6周末处死所有小鼠，观察瘤体大小变化、肺表面转移灶数、生存期等。结果 各治疗组均能抑制肿瘤增长及肺内转移，与对照组比较有统计学意义（P＜0.01），小鼠活动能力、饮食、对外界刺激的反应能力均无明显改变，生存期明显长于对照组。结论 SA脂质体介导血管抑素和／或内皮抑素基因治疗可有效地抑制Lewis肺癌移植瘤的生长、转移，生存期明显长于对照组。     

恩度联合放射线对鼻咽癌细胞裸小鼠移植瘤生物效应及机制研究

研究认为抗血管生成药物通过抑制血管新生具有抗肿瘤浸润和转移的作用,其中内皮抑素最引人关注。恩度是我国首例重组人血管内皮抑素,可诱导血管内皮细胞凋亡、抑制肿瘤新生血管形成,进而抑制细胞增殖和迁移。其对鼻咽癌的相关作用及其机制研究报道尚少见,故采用人鼻咽癌细胞(CNE-2)裸小鼠移植瘤模型观察恩度联合放射线对其生长抑制作用,并通过免疫组化技术检测细胞增殖和凋亡相关基因的表达,探讨其机制。     

重组人内皮抑素的纯化及抗肿瘤活性研究

[目的]从高效表达的基因工程菌中纯化重组人内皮抑素,对其抗肿瘤活性进行研究.[方法]经异丙基硫代-β-D-半乳糖苷(IPTG)诱导,重组人内皮抑素在大肠杆菌基因工程菌中以包涵体形式高效表达.通过凝胶层析纯化重组内皮抑素蛋白.应用鸡胚绒毛尿囊膜实验(CAM),肺癌细胞MTT试验及细胞迁移抑制实验,裸鼠皮下移植喉癌抑瘤实验、病理组织切片、免疫组化指标的测定等检测研究重组人内皮抑素的抗肿瘤活性.[结果]重组内皮抑素的复性率可达40%.重组内皮抑素在体外直接抑制肺癌细胞的增殖及迁移.用药21天,对裸鼠皮下移植喉癌的抑瘤率达到40.66%,HE染色、免疫组化及CAM实验表明其对肿瘤组织新生血管的生成有较强的抑制作用.[结论]重组人内皮抑素具有抑制肿瘤组织新生血管生成和直接抑制肿瘤细胞生长和迁移的双重抗肿瘤活性.     

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。     

重组内皮抑素的临床研究进展

重组内皮抑素能特异地抑制肿瘤血管形成,有效抑制肿瘤的生长和转移,为肿瘤的治疗提供了新途径.近几年的临床研究证实,重组内皮抑素无论单独应用还是与化疗药物联合应用均显示出了良好的抗肿瘤效果,且毒性低,安全性好,值得临床进一步深入研究和推广.本文就重组内皮抑素的临床应用进展做一综述.     

血管能抑素的应用及研究进展

血管能抑素是继血管生成抑制素、内皮抑素之后新发现的一种血管生成和肿瘤生长抑制因子。动物实验研究结果显示,血管能抑素通过抑制血管生成、阻断肿瘤生长的营养供应,从而抑制了多种肿瘤细胞的生长和转移。近年来因其具有良好的抗肿瘤血管生成活性而倍受关注,成为抗肿瘤治疗研究的新热点之一。本文就其作用机制尤其是在肿瘤治疗中的应用现状及研究进展进行综述和展望。     

血管内皮抑素对小鼠体内Lewis肺癌血管生成及转移影响的研究

目的：观察血管内皮抑素（Endostatin）对C57小鼠体内Lewis肺癌生长、血管生成及转移的影响。方法：将荷Lewis肺癌的C57鼠进行不同剂量组的内皮抑素和顺铂干预，观察肿瘤生长、移植瘤及转移瘤体内的血管内皮生长因子（VEGF）和微血管密度（MVD）的表达及转移发生率，并作统计学分析。结果：内皮抑素对鼠Lewis肺癌的生长有明显的抑制作用。内皮抑素处理组（内含400μg、300μg、200μg和200μg＋DDP组）和模型组转移率间的差异有统计学意义（P=0．0303）。内皮抑素能显著下调移植瘤及转移瘤内的VEGF。移植瘤中模型组与其余各组之间均存在显著差异（P〈0．01）；400μg组与200μg组、DDP组、联合用药组之间均存在统计学差异（P〈0．05）；300μg组与200组之间存在统计学差异（P〈0．05）；转移瘤中模型组与其余各组之间均存在统计学差异（P〈0．05）。300μg与200μg之间有统计学差异（P〈0．05），200μg与DDP组和200μg＋DDP组之间有统计学差异（P〈0．05）。原位移植瘤与肺转移肿瘤组织中VEGF表达呈正相关（r=0．977，P=0．001）。内皮抑素能显著下调移植瘤及转移瘤内和MVD。在移植瘤中400μg和300μg组肿瘤微血管密度最小，彼此无差异；200μg加DDP组肿瘤微血管密度次之，200μg组肿瘤微血管密度再次，DDP组肿瘤微血管密度在各实验组中最多，模型组肿瘤微血管密度最大。在转移瘤中400μg、300μg和200μg组微血管密度最小，这三组彼此无差异。200μg加DDP组肺转移瘤组织微血管密度次之，DDP组肺转移瘤组织微血管密度在各实验组中最多；模型组肿瘤微血管密度最大。内皮抑素可以减少肿瘤肺转移，作用程度在一定范围内与内皮抑素剂量呈正相关。结论：血管内皮抑素可以通过下调瘤组织中的VEGF和MVD抑制肿瘤生长及转移。     

Gene therapy delivery of endostatin enhances the treatment efficacy of radiation.

BACKGROUND AND PURPOSE: To evaluate whether sustained expression of mouse endostatin by adeno-associated virus (AAV)-mediated gene transfer can enhance the treatment efficacy of ionizing radiation. MATERIALS AND METHODS: Mouse endostatin was cloned into recombinant AAV (rAAV) under the control of CMV beta-actin promoter. Recombinant mouse endostatin expressed via AAV gene transfer was tested for biological activity in endothelial cells. The impact of elevated serum levels of endostatin on tumor-induced angiogenesis was evaluated using an in vivo angiogenesis assay. The anti-tumor efficacy of combining rAAV-mediated endostatin delivery with radiation was evaluated in a human colorectal tumor model (HT29). RESULTS: Recombinant mouse endostatin expressed through an AAV vector (rAAV-mEndo) inhibited endothelial cell proliferation (by 40-45%) and migration (by 22-33%). Intramuscular injection of rAAV-mEndo (1x10(9) i.u.) led to a sustained serum endostatin level of approximately 500 ng/ml. Compared to control animals this endostatin level was sufficient to inhibit tumor cell-induced vessel formation (37 vs. 28.5, P<0.05) and delay the growth of HT29 xenografts (time from 200 to 1,000 mm(3), 21 vs. 34.5 days, P<0.05). When combined with ionizing radiation, elevated serum endostatin levels significantly enhanced the time for tumors to grow from 200 to 1,000 mm(3) (radiation, 34 days; endostatin plus radiation, 50 days, P<0.05). CONCLUSION: The delivery of endostatin via rAAV vectors may provide an effective means of enhancing the anti-tumor efficacy of radiation therapy.     

Recombinant adeno-associated virus 2-mediated antiangiogenic prevention in a mouse model of intraperitoneal ovarian cancer.

PURPOSE: In the present study, we sought to determine the potential of sustained transgene expression by a single i.m. administration of recombinant adeno-associated virus 2 (rAAV) encoding angiostatin and endostatin in inhibiting i.p. ovarian cancer growth and dissemination in a preclinical mouse model. EXPERIMENTAL DESIGN: Cohorts of female athymic nude mice received either no virus or 1.2 x 10(11) particles of rAAV encoding green fluorescence protein or endostatin plus angiostatin, i.m. Three weeks later, the mice were i.p. injected with 10(6) human epithelial ovarian cancer cell line SKOV3.ip1. As a measure of effectiveness of the therapy, tumor weight, abdominal distension, ascites volume and vascular endothelial growth factor level, and tumor weight were determined. Immunohistochemistry was done to determine tumor cell apoptosis and endothelial cell proliferation following the therapy. Tumor-free survival was recorded as the end point. RESULTS: Results indicated a significant tumor-free survival (P < 0.003) following therapy with rAAV encoding endostatin and angiostatin compared with untreated or rAAV-green fluorescence protein-treated mice. Ascites volume in rAAV endostatin and angiostatin-treated mice was significantly lower than naive mice and contained less hemorrhage and tumor conglomerates. The level of vascular endothelial growth factor in the ascites of antiangiogenic vector treated mice was also significantly less compared with the untreated mice. Immunohistochemical analyses indicated increased tumor cell apoptosis and decreased blood vasculature following rAAV endostatin and angiostatin treatment. CONCLUSION: The results indicate that antiangiogenic genetic prevention from stable systemic levels of angiostatin and endostatin by i.m. administration of rAAV can be used for the treatment of i.p. ovarian cancer growth and dissemination.     

Adeno-associated virus-mediated delivery of a mutant endostatin in combination with carboplatin treatment inhibits orthotopic growth of ovarian cancer and improves long-term survival

A human ovarian cancer cell line, which migrates to mouse ovaries and establishes peritoneal carcinomatosis, was used to evaluate the cooperative effect of an antiangiogenic gene therapy combined with chemotherapy. The ovarian carcinoma cell line MA148 was genetically modified by "Sleeping Beauty" transposon-mediated delivery of DsRed2 fluorescent protein. Stable, high-level expression of DsRed protein enabled in vivo imaging of peritoneal dissemination of ovarian cancer. Both external and internal imaging, along with histopathology, showed migration of i.p. injected human ovarian cancer cell line to mouse ovaries. Using this model, we evaluated the effect of adeno-associated virus (AAV)-mediated expression of a mutant endostatin either alone or in combination with carboplatin treatment. A single i.m. injection of recombinant AAV (rAAV)-mutant human endostatin with P125A substitution (P125A-endostatin) showed sustained expression of mutant endostatin. Antiangiogenic gene therapy inhibited orthotopic growth of ovarian cancer and resulted in 33% long-term tumor-free survival. A single cycle of carboplatin treatment combined with mutant endostatin gene therapy resulted in 60% of the animals remaining tumor free for >200 days, which was significantly better than rAAV-LacZ and/or carboplatin. Combination treatment delayed tumor appearance in 40% of the animals, wherein the residual tumors were smaller in size with limited or no peritoneal metastasis. These studies suggest that AAV-mediated gene therapy of P125A-endostatin in combination with carboplatin is a useful method to inhibit peritoneal dissemination of ovarian carcinoma.     

Adeno-associated virus 2-mediated antiangiogenic cancer gene therapy: long-term efficacy of a vector encoding angiostatin and endostatin over vectors encoding a single factor

Angiogenesis is characteristic of solid tumor growth and a surrogate marker for metastasis in many human cancers. Inhibition of tumor angiogenesis using antiangiogenic drugs and gene transfer approaches has suggested the potential of this form of therapy in controlling tumor growth. However, for long-term tumor-free survival by antiangiogenic therapy, the factors controlling tumor neovasculature need to be systemically maintained at stable therapeutic levels. Here we show sustained expression of the antiangiogenic factors angiostatin and endostatin as secretory proteins by recombinant adeno-associated virus 2 (rAAV)-mediated gene transfer. Both vectors provided significant protective efficacy in a mouse tumor xenograft model. Stable transgene persistence and systemic levels of both angiostatin and endostatin were confirmed by in situ hybridization of the vector-injected tissues and by serum ELISA measurements, respectively. Whereas treatment with rAAV containing either endostatin or angiostatin alone resulted in moderate to significant protection, the combination of endostatin and angiostatin gene transfer from a single vector resulted in a complete protection. These data suggest that AAV-mediated long-term expression of both endostatin and angiostatin may have clinical utility against recurrence of cancers after primary therapies and may represent rational adjuvant therapies in combination with radiation or chemotherapy.     

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。     

Inhibition of angiogenesis and tumor progression by hydrodynamic cotransfection of angiostatin K1-3, endostatin, and saxatilin genes

In vivo expression of angiostatin and endostatin, two different types of endothelial cell growth inhibitor, have been reported to inhibit vascularization in tumor tissues, resulting in tumor growth inhibition. Recently, in vivo expression of saxatilin, a novel disintegrin purified from snake (Gloydius saxatilis) venom, was able to strongly inhibit endothelial cell proliferation and smooth muscle cell migration, resulting in tumor growth inhibition. However, the antitumor efficacy of the individual antiangiogenic molecules expressed in vivo was not sufficiently potent to induce tumor regression in animal models. Therefore, in this study, we have systemically examined how combinational transfer of angiostatin, endostatin, and saxatilin genes affects neovascularization in tumor tissues and tumor progression in a mouse model. In Matrigel-implanted mice, cotransfection with plasmids encoding angiostatin K1-3 (pFLAG-Angio K1/3), endostatin (pFLAG-Endo), and saxatilin (pFLAG-Sax) resulted in the most effective inhibition of angiogenesis. In addition, hydrodynamic cotransfection of the three genes induced more inhibition of B16BL6 melanoma growth and pulmonary metastasis than other combinations of transfected genes. Compared with the empty vector-treated control group, cotreatment with the three plasmids reduced B16BL6 tumor growth by 89% and pulmonary metastasis by 90%. These results provide additional evidence supporting the combined systemic expression of antiangiogenic factors, such as angiostatin K1-3, endostatin, and saxatilin, as an alternative procedure for antiangiogenic cancer therapy.     

Suppression of lung tumor growth and metastasis in mice by adeno-associated virus-mediated expression of vasostatin.

PURPOSE: Angiogenesis inhibitors have strong therapeutic potential as antitumor agents in suppressing tumor growth and metastatic progression. Vasostatin, the N-terminal domain of calreticulin, is a potent angiogenesis inhibitor. In this study, we determined the effectiveness of vasostatin delivered by recombinant pseudotype adeno-associated virus 2/5 (rAAV2/5-VAS) as a gene therapy approach for lung cancer treatment. EXPERIMENTAL DESIGN: We used rAAV2/5 to deliver vasostatin intratumorally or systemically in different mouse lung tumor models--subcutaneous, orthotopic xenograft, and spontaneous metastasis lung tumor models. The therapeutic efficacy of rAAV2/5-VAS was determined by monitoring tumor volume, survival rate, and degree of neovascularization after treatment in these models. RESULTS: Mice bearing subcutaneous tumor of rAAV2/5-VAS pretreated Lewis lung carcinoma cells showed >50% reduction in primary tumor volume and reduced spontaneous pulmonary metastases. The tumor-suppressive action of rAAV2/5-VAS in subcutaneous human lung tumor A549 xenograft correlated with a reduced number of capillary vessels in tumors. In the orthotopic xenograft model, rAAV2/5-VAS suppressed metastasis of A549 tumors to mediastinal lymph nodes and contralateral lung. Furthermore, treatment of immunocompetent mice in the spontaneous lung metastases model with rAAV2/5-VAS after primary tumor excision prolonged their median survival from 21 to 51.5 days. CONCLUSION: Our results show the effectiveness of rAAV2/5-VAS as an angiogenesis inhibitor in suppressing tumor growth during different stages of tumor progression, validating the application of rAAV2/5-VAS gene therapy in treatment against lung cancer.