血管抑制素和内皮抑制素的协同作用

血管抑制素和内皮抑制素是内源性的血管生成抑制剂,对肿瘤有明显的抑制作用,具有良好的应用前景,近年来,对于它们之间协同作用的研究已经成为新的热点。本文除对两者做了结构、性质、功能等方面的一般性介绍外,重点介绍了它们之间的协同作用。与血管抑制素和内皮抑制素单体的作用相比,它们之间的联合使用具有更强的抑制血管生成和肿瘤生长的作用。     

重组人血管内皮抑制素对荷瘤裸小鼠的治疗作用

观察了重组人血管内皮抑制素 (YH - 16 )对人肝癌Be174 0 2 细胞系、人宫颈癌Hela细胞系和人胃癌SGC790 1细胞系在裸小鼠体内的抑制作用。试验采用每日尾静脉注射给药 ,YH - 16的剂量分别为 1.5、0 .75、0 .4mg/kg ,共给药 14次。结果表明YH - 16对上述三种人癌细胞系有明显抑制作用 ,其抑制率Be174 0 2 为 45 .6 7、43.0 8和 40 .0 0 % ;Hela为 40 .70、30 .15和2 4.12 % ;SGC790 1为 5 1.89、44 .34和 35 .85 %。YH— 16各给药组动物健康状况良好 ,无明显毒副反应     

血管内皮抑制素的研究进展

实体瘤当生长到一定的体积时必然伴随着血管生成。 1971年 ,Ｆｏｌｋｍａｎ[1] 最早提出肿瘤生长是血管依赖性的。他认为肿瘤血管生成是肿瘤生长和转移的形态学基础。它不仅向肿瘤提供充足的营养 ,同时也向机体输出肿瘤细胞 ,导致肿瘤细胞的恶性生长和转移。因此 ,Ｆｏｌｋｍａｎ[2 ] 提出抗血管疗法 ,设想抑制肿瘤血管生成 ,使肿瘤细胞因缺血、缺氧而大部分死亡 ,从而可延缓晚期肿瘤生长 ,延长病人带瘤生存期 ,并可抑制亚临床转移灶生长 ,推迟复发。这一设想为越来越多的证据所支持 ,并使这一领域成为肿瘤研究的热点及肿瘤治疗的新策略。人…     

重组人血管内皮抑制素对荷瘤裸小鼠的治疗作用

观察了重组人血管内皮抑制素(YH-16)对人肝癌Bel7402细胞系、人宫颈癌Hela细胞系和人胃癌SGC7901细胞系在裸小鼠体内的抑制作用。试验采用每日尾静脉注射给药，YH-16的剂量分别为1.5、0.75、0.4mg/kg，共给药14次。结果表明YH-16对上述三种人癌细胞系有明显抑制作用，其抑制率Bel7402为45.67、43.08和40.00％；Hela为40.70、30.15和24.12％；SGC7901为51.89、44.34和35.85％。YH-16各给药组动物健康状况良好，无明显毒副反应。     

重组人血管内皮抑制素可抑制兔耳增生性瘢痕

背景：增生性瘢痕局部血流量明显增高,微血管密度明显高于萎缩期及成熟的正常瘢痕。 目的：观察重组人血管内皮抑制素对新西兰大耳兔兔耳增生性瘢痕的抑制作用及其对瘢痕中血管内皮生长因子的作用。 方法：建立兔耳增生性瘢痕动物模型。随机分为2组,实验组局部注射重组人血管内皮抑制素,对照组局部注射生理盐水。 结果与结论：药物干预后30 d实验组兔耳瘢痕较对照组颜色变淡、质地柔软、厚度薄。苏木精-伊红染色实验组真皮层较对照组薄,单位面积内成纤维细胞数较对照组少,呈平行排列,毛细血管管径较对照组细;实验组瘢痕增生指数显著低于对照组(P < 0.01)。实验组瘢痕组织血管内皮生长因子阳性表达量显著低于对照组(P < 0.01)。说明重组人血管内皮抑制素注射液可能通过降低血管内皮生长因子蛋白的表达,从而抑制兔耳增生性瘢痕组织增生。     

重组人血管内皮抑制素对小鼠胃癌c-Myc,bFGF表达的影响

目的 通过构建荷胃癌小鼠模型研究重组人血管内皮抑制素对癌基因c-Myc和成纤维细胞生长因子(bFGF)表达的影响及可能机制.方法 20只小鼠皮下注射MFC小鼠前胃癌细胞从而构建小鼠胃癌移植瘤模型,后随机分成两组,每组10只,观察组腹腔注射0.8mg/kg重组人血管内皮抑素,对照组腹腔注射等量生理盐水.每天观察小鼠的一般情况,每3天测量肿瘤体积,绘制肿瘤体积生长曲线并对两实验组肿瘤体积进行比较;应用RT-PCR、Western bolt及免疫组织化学法检测两组小鼠肿瘤组织中c-Myc和bFGF的表达,免疫组织化学染色法检测肿瘤组织微血管密度(MVD)的表达,并通过Spearman分析c-Myc和bFGF两者的相关性.结果 在用药的过程中,对照组小鼠随着移植瘤结节的增大而发生行为明显改变,但观察组小鼠精神、反应、活动及进食饮水均如初,未见明显不良反应.治疗后第9d开始,观察组小鼠移植瘤体积明显小于对照组(P＜0.05),在第21 d最明显(P＜0.01).RT-PCR检测显示e-Myc,bFGF mRNA相对表达量明显低于对照组(P＜0.05);Western blot检测显也显示观察组c-Myc,bFGF蛋白的表达量明显低于对照组(P＜0.05);免疫组织化学检测显示c-Myc,bFGF阳性细胞数均明显低于对照组(P＜0.05),c-Myc,bFGF平均灰度值明显高于对照组(P＜0.05);对照组平均MVD为9.2±1.3,观察组为2.9±1.0,两组之间差异亦具有统计学差异(P＜0.05).且Spearman分析得出小鼠胃移植瘤中e-Myc和bFGF mRNA存在正相关(r=0.853,P=0.000).结论 重组人血管内皮抑制素通过抑制胃癌组织c-Myc,bFGF基因表达和血管生成.     

血管抑制素和内皮抑制素在大肠杆菌中的融合表达与鉴定

血管抑制素（AS）和内皮抑制素（ES）是两种具有较强活性的内源性血管抑制剂，联合应用具有协同作用。本研究通过大肠杆菌表达AS-ES融合蛋白，观察其对血管生成的抑制作用。首先用RT-PCR方法分别获得AS和ES基因，通过基因拼接获得融合基因，构建了含有该融合基因的原核表达质粒——pET-42（b）／AS-ES。表达菌株经IPTG诱导后目的蛋白以包涵体形式表达，表达量为14％，分子量约65KD。Western blotting检测表明表达产物可分别与AS和ES抗体产生特异的免疫反应。经复性、肝素亲和层析柱纯化的表达产物对鸡胚尿囊膜血管有明显抑制作用。本研究获得了AS、ES在大肠杆菌中的融合表达．目的蛋白具有特异的免疫反应性和血管抑制活性。     

内皮抑制素的研究进展

肿瘤生长依赖新生血管的形成。通过抑制血管内皮细胞 ,切断肿瘤生长所需的营养途径 ,阻断新生血管形成 ,从而达到抑制肿瘤生长的目的。 Endostatin(内皮抑制素 )具有抑制肿瘤血管生成和肿瘤转移最强烈、最特异的生物学活性 ,且不产生耐药 ,是目前发现最为理想的血管生成抑制因子     

温度和pH对重组人血管内皮抑制素脱酰胺的影响

目的利用液质联用技术研究温度和pH对重组人血管内皮抑制素中天冬酰胺(Asn)脱酰胺过程的影响。方法在不同的温度和pH值条件下,对重组人血管内皮抑制素样品溶液进行孵化,酶解处理后利用液质联用技术识别发生脱酰胺的多肽并测定其含量随样品放置温度和pH条件的变化。结果质谱分析结果表明酶解产物中多肽SVWHGSDPNGR序列中的Asn发生了脱酰胺化,Asn残基生成了天冬氨酸和异天冬氨酸残基,其他位置的Asn均未检测出明显的脱酰胺化;实验获得了不同温度和pH条件下重组人血管内皮抑制素中Asn脱酰胺化的速率及动力学常数;Asn脱酰胺过程随着温度和溶液pH值的升高,Asn脱酰胺速率上升。结论重组人血管内皮抑制素中天冬酰胺的脱酰胺过程受储存温度和pH值影响,研究药用蛋白质在不同条件下的脱酰胺过程对于筛选合适的储存条件、减少活性损失具有重要意义。     

环磷酰胺节拍化疗联合重组人血管内皮抑制素对非小细胞肺癌移植瘤裸鼠的影响及其机制研究

 目的：本研究观察环磷酰胺（cyclophosphamide, CPA）节拍化疗（metronomic chemotherapy,MET）联合重组人血管内皮抑制素（recombinant human endostatin,Endostar）用于非小细胞肺癌（non-small-cell lung cancer, NSCLC）维持治疗的疗效和抗血管形成机制,探索一种新的晚期NSCLC维持治疗模式。方法：建立人肺腺癌细胞系A549裸鼠皮下移植瘤模型,以最大耐受剂量（maximum tolerated dose,MTD）CPA化疗4个周期后随机分组,转换成以下治疗：生理盐水（control组）、MET CPA维持治疗（MET CPA组）、Endostar维持治疗（Endo组）和MET CPA联合Endostar维持治疗（MET CPA+Endo组）。记录模型动物的移植瘤体积和总生存期。流式细胞术测定外周血循环内皮细胞（circulating endothelial cells, CECs）和存活的CECs（viable CECs）,共聚焦显微镜测定肿瘤微血管密度（microvessel density,MVD）和管周细胞覆盖率。结果：在维持治疗第6周时,MET CPA和Endo组肿瘤体积显著小于control组（P<0.05）,MET CPA+Endo组肿瘤体积又显著小于单药维持治疗组（P<0.05）。MET CPA和Endo组小鼠生存期显著长于control组（P<0.05）,MET CPA+Endo组小鼠生存期则进一步延长（P<0.05）。与control组比较,MET CPA和Endo组显著下调了外周血CECs和存活CECs水平（P<0.01）,并降低了移植瘤MVD（P<0.01）。Endo组显著下调了移植瘤血管管周细胞覆盖率（P<0.05）。在上述血管形成相关指标上,MET CPA+Endo组又显著低于单药维持治疗组（P<0.05）。结论：MET CPA联合Endostar用于NSCLC肺腺癌维持治疗能延缓移植瘤生长,改善荷瘤鼠生存期,这可能与联合用药具有抗血管形成的增效作用有关。本研究为晚期NSCLC维持治疗提供了新的治疗思路,值得进行临床研究。     

Inhibitory effects of roxithromycin on tumor angiogenesis, growth and metastasis of mouse B16 melanoma cells

We examined the effects of roxithromycin, a 14-membered ring macrolide antibiotic, on tumor angiogenesis, tumor growth and metastasis of mouse B16BL6 melanoma cells. The inhibitory effect of roxithromycin on angiogenesis using mouse dorsal air sac model was dose-dependent, and 100 mg/kg of roxithromycin administered intraperitoneally twice a day reduced the dense capillary network area to about 20% of the control. Administration of roxithromycin histologically reduced the development of microvessels and mononuclear cell infiltration. In vivo tumor growth studies demonstrated that intraperitoneal administration of roxithromycin at 20 mg/kg/day and 50 mg/kg/day reduced tumor size of B16BL6 melanoma to about 56% and 33% (experiment 1), 71% and 48% (experiment 2) of that in the respective controls. Roxithromycin also significantly inhibited pulmonary metastasis of B16BL6 cells in a spontaneous system. The inhibitory activities of roxithromycin on angiogenesis, tumor growth and metastasis were compared with those of a potent angiogenesis inhibitor, TNP-470. These data demonstrated that roxithromycin has potent antiangiogenic and antitumor effects and might have possible therapeutic applications.     

Mast cells impair melanoma cell homing and metastasis by inhibiting HMGA1 secretion

Metastatic disease is the major cause of death from cancer. From the primary tumour, cells remotely prepare the environment of the future metastatic sites by secreted factors and extracellular vesicles. During this process, known as pre-metastatic niche formation, immune cells play a crucial role. Mast cells are haematopoietic bone marrow-derived innate immune cells whose function in lung immune response to invading tumours remains to be defined. We found reduced melanoma lung metastasis in mast cell-deficient mouse models (Wsh and MCTP5-Cre-RDTR), supporting a pro-metastatic role for mast cells in vivo. However, due to evidence pointing to their antitumorigenic role, we studied the impact of mast cells in melanoma cell function in vitro. Surprisingly, in vitro co-culture of bone-marrow-derived mast cells with melanoma cells showed that they have an intrinsic anti-metastatic activity. Mass spectrometry analysis of melanoma-mast cell co-cultures secretome showed that HMGA1 secretion by melanoma cells was significantly impaired. Consistently, HMGA1 knockdown in B16-F10 cells reduced their metastatic capacity in vivo. Importantly, analysis of HMGA1 expression in human melanoma tumours showed that metastatic tumours with high HMGA1 expression are associated with reduced overall and disease-free survival. Moreover, we show that HMGA1 is reduced in the nuclei and enriched in the cytoplasm of melanoma metastatic lesions when compared to primary tumours. These data suggest that high HMGA1 expression and secretion from melanoma cells promote metastatic behaviour. Targeting HMGA1 expression intrinsically or extrinsically by mast cells actions reduce melanoma metastasis. Our results pave the way to the use of HMGA1 as anti-metastatic target in melanoma as previously suggested in other cancer types.     

重组人内皮抑素腺病毒抗肿瘤实验研究

目的肿瘤生长具有血管依赖性。内皮抑素为胶原X羧基末端裂解片段,是重要的内源性血管抑制因子。实验中利用重组人内皮抑素腺病毒(recombinanthumanendostatinadenorirus,Ad-hEndo)在肿瘤局部给药以探索其抗血管基因治疗的可行性。方法以Ad-hEndo感染体外培养肿瘤细胞,观察重组蛋白表达及其对培养的血管内皮细胞的抑制效应;在裸鼠A549肺癌模型中瘤内注射重组病毒,观察肿瘤抑制效应、剂量依从效应和毒副反应。结果不同感染复数的Ad-hEndo感染肿瘤细胞均表达重组内皮抑素蛋白,并能抑制血管内皮细胞的生长。动物实验中Ad-hEndo治疗组肿瘤体积及肺转移结节数明显低于对照组,且转移数与治疗剂量负相关。结论以腺病毒为载体的肿瘤局部血管基因治疗能抑制肿瘤新生血管形成进而有效抑制肿瘤生长和转移,其效应具有剂量依从性。     

Anti-tumor immunoglobulin M increases lung metastasis in an experimental model of malignant melanoma

Cancer metastasis involves distinct steps that depend on complicated tumor–host interactions. The hematogenous dissemination of tumor cells may be facilitated by factors that promote the arrest and adherence of cancer cells in capillaries. We examined whether anti-tumor monoclonal immunoglobulin M (IgM) antibodies promoted the hematogenous dissemination of B16 melanoma cells in syngeneic mice. IgM monoclonal antibodies were generated that selectively bind to B16 melanoma cells as compared to syngeneic fibroblasts, lymphocytes or Lewis lung carcinoma cells. Incubation of B16-BL6 or B16-F0 melanoma cells with these IgM anti-tumor antibodies significantly increased the number of lung colonies as compared with control antibodies. Moreover, intraperitoneal injection of specific antibody also significantly increased lung colonization. All anti-tumor antibodies promoted the aggregation of B16 melanoma cells. A chemically generated immunoglobulin G (IgG)-like fragment of an anti-tumor IgM antibody displayed greatly reduced tumor aggregation and, in contrast to intact IgM, did not significantly increase lung colonization of B16 melanoma cells. Neither intact IgM nor the IgG-like fragment enhanced the in vitro invasiveness of B16 melanoma cells across Matrigel-coated membranes. Our results, therefore, suggest that besides their beneficial anti-tumor effects, anti-tumor IgM antibodies may also promote the hematogenous dissemination of cancer cells. This revised version was published online in July 2006 with corrections to the Cover Date.     

Molecular determinants of human uveal melanoma invasion and metastasis

The molecular analysis of cancer has benefited tremendously from the sequencing of the human genome integrated with the science of bioinformatics. Microarray analysis technology has the potential to classify tumors based on the differential expression of genes. In the current study, a collaborative, multidisciplinary approach was utilized to study the molecular determinants of human uveal melanoma invasion and metastasis. Uveal melanoma is considered the most common primary intraocular cancer in adults, resulting in the death of approximately 50% of patients affected. Unfortunately, at the time of diagnosis, many patients already harbor microscopic metastases, thus underscoring a critical need to identify prognostic markers indicative of metastatic potential. The investigative strategy consisted of isolating highly invasive vs. poorly invasive uveal melanoma cells from a heterogeneous tumor derived from cells that had metastasized from the eye to the liver. The heterogeneous tissue explant MUM-2 led to the derivation of two clonal cell lines: MUM-2B and MUM-2C. Further morphological and functional analyses revealed that the MUM-2B cells were epithelioid, interconverted (expressing mesenchymal and epithelial phenotypes) highly invasive, and demonstrated vasculogenic mimicry. The MUM-2C cells were spindle-like, expressed only a vimentin mesenchymal phenotype, poorly invasive, and were incapable of vasculogenic mimicry. The molecular analysis of the MUM-2B vs. the MUM-2C clones resulted in the differential expression of 210 known genes. Overall, the molecular signature of the MUM-2B cells resembled that of multiple phenotypes – similar to a pluripotent, embryonic-like genotype. Validation of select genes that were upregulated and down-regulated was conducted by semiquantitative RT-PCR measurement. This study provides a molecular profile that will hopefully lead to the development of new molecular targets for therapeutic intervention and possible diagnostic markers to predict the clinical outcome of patients with uveal melanoma. This revised version was published online in July 2006 with corrections to the Cover Date.     

Delivery of endostatin in experimental cancer therapy

Endostatin, the 20 kDa C-terminal fragment of collagen XVIII, has been shown to be an effective inhibitor of tumour angiogenesis and growth in different experimental systems and is currently in Phase II/III clinical trials. One challenging aspect of anti-angiogenic treatment is the mode of delivery of the active compound. In this paper we review some of the basic knowledge of endostatin and look specifically into the different possible ways in which endostatin may be administered.     

Inhibition of experimental metastasis and cell adhesion of murine melanoma cells by chondroitin sulfate-derivatized lipid, a neoproteoglycan with anti-cell adhesion activity

Chondroitin sulfate dipalmitoylphosphatidylethanolamine (CS-PE), when immobilized onto substratum, inhibited the adhesion of B16F10 mouse melanoma cells to fibronectin-coated dishes (anti-adhesion activity). CS-PE showed the most potent anti-adhesion activity for the melanoma cells among various GAG-PEs. CS-PE also inhibited the adhesion of B16F10 cells to Matrigel and the invasion of the cells into Matrigel. In the in vivo system of experimental metastasis, administration of B16F10 cells with CS-PE into C57BL/6 mice significantly inhibited lung metastasis. The inhibition degree of CS or hyaluronic acid-PE was lower than CS-PE. CS-PE administered intravenously into mice before the injection of B16F10 cells also inhibited metastasis. Pretreatment of B16F10 cells with CS-PE caused some but a lower degree of inhibition. When CS-PE was injected intravenously into mice, more binding in the lung was found than when CS was injected. CS-PE but not CS inhibited the retention in the lung of fluorochrome-labeled B16F10 cells when injected intravenously into mice. Since there was no significant effect of CS-PE on the viability and growth of B16F10 cells, the results suggest that CS-PE immobilized onto the subendothelial matrix may prevent melanoma cells from adhering to the subendothelial substrata of lung capillaries and inhibit subsequent invasion processes of metastasis.     

Pleural metastasis from auricular melanoma: A brief report

Primary auricular melanoma is rarely reported. Approximately, it accounts for 1% to 4% of all cutaneous melanoma. Early literature suggested that melanoma of the ear is more aggressive than other melanomas, with a propensity for spreading to both regional lymph nodes and distant sites. Here, we present a case of cytological pleural metastasis from auricular melanoma in a 43‐year‐old woman. Immunohistochemical staining showed that the tumors cells were positive for S‐100 protein and Melan‐A. The mutation of the v‐raf murine sarcoma viral oncogene homolog B (BRAF)^V600E was demonstrated on Sanger sequencing. To our knowledge, this is the first report describing the cytomorphology of metastatic auricular melanoma in pleural effusion.     

Human melanoma invasion and metastasis enhancement by high expression of aminopeptidase N/CD13

Aminopeptidase N/CD13 is a Zn²⁺-dependent exoprotease present on the cell surface as a transmembrane protein. Our previous studies using aminopeptidase inhibitors and antibodies demonstrated that aminopeptidase N is involved in the degradation and invasion of the extracellular matrix (ECM) by metastatic tumor cells. In the present study we transfected human A375M melanoma cells with eukaryotic plasmid expression vectors that contained full length cDNA of aminopeptidase N/CD13 and examined their characteristics. The transfectants that expressed extremely high levels of aminopeptidase N/CD13 degraded type IV collagen and invaded ECM more actively than the parental and control vector-transfected cells. Furthermore, the aminopeptidase N/CD13-transfected A375M cells had significantly augmented lung colonizing potential in nude mice. The results show that the aminopeptidase N/CD13 plays an active role in degradation and invasion of ECM and may be involved in the molecular mechanisms of blood-borne metastasis.