基质金属蛋白酶-9与骨肉瘤鸡胚移植瘤血管生成的关系

目的 建立人骨肉瘤鸡胚移植瘤模型，探讨基质金属蛋白酶-9（Matrix Metalloproteinase-9，MMP-9）在移植瘤模型中的表达及其与肿瘤血管生成的关系。方法 人骨肉瘤细胞接种于鸡胚绒毛尿囊膜（chick embryo chorioallantoic membrane，CAM），建立人骨肉瘤鸡胚移植瘤模型，观察移植瘤生长特性和生物学性状；采用免疫组化法检测移植瘤组织中MMP-9的表达及微血管密度（microvessel density，MVD）。结果 建立了人骨肉瘤鸡胚移植瘤模型，移植瘤具有较强的诱导血管生成作用，骨肉瘤鸡胚移植瘤中，MMP-9的表达水平与MVD呈显著性正相关（P〈0．001，r=0．855）。结论 人骨肉瘤鸡胚移植瘤模型易于复制、操作简单、适用范围广，是研究骨肉瘤诱导血管生成的较好动物模型；人骨肉瘤鸡胚移植瘤中MMP-9表达水平与骨肉瘤诱导的血管生成密切相关。     

Ⅴ型腺病毒纤维蛋白基因真核表达质粒的构建及表达

目的：克隆VEGF受体sFET-1片段1-3区，并使之在原核中进行表达以探讨其对内皮细胞增殖和血管生成方面的抑制作用。方法：提取脐静脉血管内皮细胞总RNA，克隆sFLT-1基因1-3区，并使之在QIA表达系统进行表达，Ni^2 -Sepha-rose6B亲和层析纯化，复性，应用MTT和鸡胚绒毛尿囊膜血管生成模，探讨重组蛋白对血管生成的抑制作用。结果；该表达系统能表达sFLT-1基因片段，表达量低，纯化后，经复性，sFLT-1具有VEGF特异结合功能，1μg重组sFLT-1蛋白能抑制10ngVEGF诱导的脐静脉内皮细胞增殖和20ngVEGF诱导的鸡胚绒毛尿囊膜血管生成，结论：sFLT-1基因片段能在QIA表达系统中得到表达，复性后重组蛋白具有与VEGF结合和拮抗VEGF生物学活性的功能。     

人血管抑制因子Vasostatin短片段（120 180 aa）的克隆、表达及功能研究

目的：利用基因工程技术，克隆并表达人血管抑制因子Vasostatin120180 aa功能区片段，探讨其对鸡胚绒毛尿囊膜新生血管抑制的作用。方法：采用PCR技术扩增人血管抑制因子Vasostatin120180 aa功能区基因，并利用pQE30原核表达系统诱导表达Vasostatin120180aa，经镍金属螯合层析法纯化，通过鸡胚绒毛尿囊膜实验验证其抑制新生血管生成的作用。结果：PCR扩增出了长度为180 bp的Vasostatin120180 aa功能区基因，后经pQE30原核表达系统表达并纯化出Vasostatin120180 aa，SDSPAGE显现一条约8 kD的阳性条带，Vasostatin120180aa可显著抑制鸡胚绒毛尿囊膜新生血管的生成。结论：原核表达的Vasostatin120180 aa功能区片段具有生物学活性，可明显抑制鸡胚绒毛尿囊膜新生血管的生成，在一定范围内呈量效依赖性。     

人血管抑制因子Vasostatin短片段(120-180 aa)的克隆、表达及功能研究

目的:利用基因工程技术,克隆并表达人血管抑制因子Vasostatin120-180aa功能区片段,探讨其对鸡胚绒毛尿囊膜新生血管抑制的作用.方法:采用PCR技术扩增人血管抑制因子Vasostatin120-180aa功能区基因,并利用pQE30原核表达系统诱导表达Vasostatin120-180aa,经镍金属螯合层析法纯化,通过鸡胚绒毛尿囊膜实验验证其抑制新生血管生成的作用.结果:PCR扩增出了长度为180 bp的Vasostatin120-180aa功能区基因,后经pQE30原核表达系统表达并纯化出Vasostatin120-180aa,SDS-PAGE显现一条约8 kD的阳性条带,Vasostatin120-180aa可显著抑制鸡胚绒毛尿囊膜新生血管的生成.结论:原核表达的Vasosta-tin120-180aa功能区片段具有生物学活性,可明显抑制鸡胚绒毛尿囊膜新生血管的生成,在一定范围内呈量效依赖性.     

人骨肉瘤鸡胚移植瘤模型的生物学特性研究

目的:建立人骨肉瘤鸡胚移植瘤模型,研究其形态学及生物学特性。方法:将人骨肉瘤细胞接种于鸡胚绒毛尿囊膜(chick embryo chorioallantoic membrane,CAM),观察影响骨肉瘤鸡胚移植瘤成活的因素、移植瘤生长特性、形态学特征和生物学性状。结果:建立了人骨肉瘤鸡胚移植瘤模型。移植瘤易于生长,具有较强的血管诱导作用。光镜下移植瘤具有与人骨肉瘤类似的组织结构。结论:该模型易于复制,能动态观察骨肉瘤诱导的血管生成过程,可用于骨肉瘤的生物学行为研究、药物筛选等领域。     

高通量肿瘤血管新生研究技术平台的建立

目的：建立高通量肿瘤血管新生研究的体内外技术平台。方法：以VEGF,hFGF为血管新生因子,以Thalidomide血管新生抑制因子对体外培养的血管内皮细胞系（RF／6A,SVEC）应用MTT试验,迁移试验,浸润试验和管状形成试验检测内皮细胞的各种特性;体内实验以鸡胚C57BL／6小鼠为实验动物检测血管新生因子和抑制因子在体内对血管新生的影响。结果：VEGF和bFGF在体内和体外均有促进血管新生的作用,体外试验表明VEGF和bFGF对内皮细胞的增殖活性,迁移、浸润和管状结构形成均有明显的促进作用（P＜0．01）;小鼠体内栓子试验和鸡胚绒毛尿囊膜试验结果均表明,VEGF和b FGF对体内血管新生同样具有促进或刺激作用,和阴性对照组相比主要表现在新生血管密度高、管腔大、管壁完整等;而Thalidomide在体内外均有明显抑制血管新生的作用。结论：本技术平台是一个高通量、经济、简便易行、实用的血管新生研究技术平台。     

去甲斑蝥素对人乳腺癌血管生成的抑制作用

背景与目的:去甲斑蝥素(norcantharidin,NCTD)具有抑瘤作用,本文研究它对血管生成的作用,以及对人乳腺癌MCF-7细胞鸡胚绒毛尿囊膜(chorioallantoci membrane,CAM)移植肿瘤血管生成的影响。方法:①建立鸡胚绒毛尿囊膜模型,将明胶海绵载体置于两条前卵黄静脉间血管的CAM上,将不同剂量的NCTD加在载体上,以生理盐水对照,观察记录载体周围血管分支点数,计算血管生成抑制率。②建立人乳腺癌MCF-7细胞鸡胚移植模型。给予不同剂量的NCTD20μl[72、36、18μg/(胚.d)],以生理盐水对照,计算血管生成抑制率。结果:NCTD对以明胶海绵为载体的鸡胚绒毛尿囊膜血管生成具抑制作用,72、36、18、9μg NCTD组血管生成抑制率分别为77.7%、62.9%、50.6%及33.0%,并且呈剂量依赖性。移植肿瘤可诱发血管生成,NCTD能明显抑制MCF-7鸡胚移植肿瘤血管生成,72、36、18μg NCTD对移植瘤诱导的血管抑制率分别为66.2%、39.3%和22.8%。结论:NCTD能明显抑制鸡胚绒毛尿囊膜新生血管的生成,同时能抑制人乳腺癌MCF-7细胞鸡胚移植肿瘤的血管生成。其抗肿瘤作用与抑制肿瘤血管生成有关。     

血管内皮细胞生长因子VEGF与唾液酸化Lewis-X抗原在骨肉瘤中的表达及意义的研究

骨肉瘤是一种好发于青少年的高度恶性肿瘤,研究表明骨肉瘤的术后复发和转移是影响患者预后的主要因素.肿瘤血管的生长无疑是肿瘤发生侵袭和转移的重要因素.血管对于肿瘤的生长起巨大的作用,新生的血管的形成增殖可以使骨肉瘤迅速生长及向远处转移.许多血管生长因子在肿瘤中均有表达,介导血管新生.肿瘤的生长、侵袭和转移是血管生成依赖性的,人类的许多恶性肿瘤包括骨肉瘤都发现有高水平的血管内皮细胞生长因子(vascular endothelial growth factor,VEGF).     

胃癌组织中VEGF、bFGF表达及与微血管计数的关系

肿瘤的生长和转移均需血管生长,血管生成与促血管生成因子有关。主要促血管生成因子有：血管内皮生长因子（vascular endothelial growth factor,VEGF)、碱性成纤维细胞生长因子（basic fibroblast growth factor,bFGF）和血小板衍生生长因子（platelet derived growth factor,PDGF),近年研究发现VEGF、bFGF和PDGF表达与恶性肿瘤血管生成、生物学行为及预后有关。作者应用免疫组化研究胃癌组织中VEGF、bFGF表达及与微血管计数（microvessel coumt,MVC)的关系。     

胃癌组织中VEGF、bFGF表达及与微血管计数的关系

肿瘤的生长和转移均需血管生成,血管生成与促血管生成因子有关.主要促血管生成因子有:血管内皮生长因子(vascular endothelial growth factorVEGF)、碱性成纤维细胞生长因子(basic fibroblast growth factorbFGF)和血小板衍生生长因子(platelet derived growth factorPDGF).近年研究发现VEGF、bFGF和PDGF表达与恶性肿瘤血管生成、生物学行为及预后有关.作者应用免疫组化研究胃癌组织中VEGF、bFGF表达及与微血管计数(microvessel countMVC)的关系.     

Human chondrosarcoma secretes vascular endothelial growth factor to induce tumor angiogenesis and stores basic fibroblast growth factor for regulation of its own growth.

Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are well-known factors that induce neovascularization in many tumors. The molecular mechanisms that regulate tumor angiogenesis in human chondrosarcoma are not clear. We assessed in this work the angiogenic activities of a human chondrosarcoma cell line (OUMS-27) in vivo and determined the efficacies of angiogenic factors derived from OUMS-27 cells on human umbilical vein endothelial cells (HUVECs) in vitro. Tumor xenografts induced an increase in the formation of neovessels, but the distributions of Ki-67 antigen, VEGF and bFGF were unaffected. We also demonstrated that OUMS-27 cells secreted VEGF(165) into the culture medium and that it was the maximal angiogenic factor to stimulate endothelial proliferation and migration in chondrosarcoma. Anti-VEGF antibodies induced an approximately 70% inhibition of these responses of HUVECs, but did not have any effect on OUMS-27 cells. Anti-bFGF antibodies suppressed not only the activities of HUVECs but also the growth of tumor cells in vitro. We indicate that angiogenesis is principally elicited by VEGF(165) and that tumorigenesis is mainly regulated by bFGF stored in the extracellular matrix of OUMS-27 cells. The present study may offer the availability of combination therapies for inhibition of VEGF and bFGF action on vascular endothelial cells and chondrosarcoma cells, respectively.     

乌骨藤制剂对人肝癌细胞株ＶＥＧＦ和ｂＦＧＦ表达的影响

目的　探讨乌骨藤制剂对人肝癌细胞株ＶＥＧＦ和ｂＦＧＦ表达的影响及其抗肿瘤的作用机制。方法　采用ＥＬＩＳＡ法和细胞免疫化学ＳＰ染色法观察不同浓度的乌骨藤制剂作用人肝癌Ｂｅｌ-７４０２细胞２４ｈ、４８ｈ后ＶＥＧＦ和ｂＦＧＦ的表达。结果　不同浓度的乌骨藤制剂（１０ｍｇ／ｍｌ、２０ｍｇ／ｍｌ、４０ｍｇ／ｍｌ）作用Ｂｅｌ-７４０２细胞后,ＶＥＧＦ和ｂＦＧＦ表达均随药物浓度的增加逐渐减少,与阴性对照组相比有显著性差异。结论　乌骨藤制剂能够明显抑制人肝癌Ｂｅｌ-７４０２细胞ＶＥＧＦ和ｂＦＧＦ的表达,提示乌骨藤制剂抗肝癌的作用机制可能与抑制肿瘤血管生成密切相关。     

VEGF,bFGF and EGF in the angiogenesis of human melanoma xenografts

Vascular endothelial growth factor (VEGF) is a major inducer of angiogenesis in tumors. Basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) have both been shown to interact with VEGF. The involvement of VEGF, bFGF and EGF in melanoma angiogenesis was investigated here. Four human melanoma cell lines (A-07, D-12, R-18, U-25) were included in the study. Angiogenesis was quantified by scoring of tumor-oriented capillaries following intradermal cell inoculation in BALB/c nu/nu mice. VEGF, bFGF and EGF expression and secretion were investigated by Western blotting and enzyme-linked immunosorbent assay, respectively. Immunohistochemistry of xenografts grown intradermally was used to reveal VEGF and bFGF localization in vivo. The rate of angiogenesis differed substantially among the melanoma lines; the sequence from a high to low rate of angiogenesis was: A-07, D-12, R-18, U-25. A-07, which induced the highest rate of angiogenesis, showed a higher rate of VEGF secretion, stronger VEGF staining by immunohistochemistry and higher bFGF expression than the other lines. U-25, which induced the lowest rate of angiogenesis, showed a higher rate of VEGF secretion than D-12 and R-18. A-07 was the only line that showed detectable bFGF secretion, and R-18 was the only line that showed detectable EGF secretion. VEGF is probably important in the angiogenesis of melanomas. However, heterogeneity in rate of angiogenesis among melanomas cannot be attributed to heterogeneity in rate of secretion of VEGF, bFGF and/or EGF only. Int. J. Cancer 76:836–841, 1998.© 1998 Wiley-Liss, Inc.     

Vascular endothelial growth factor, interleukin 8, platelet-derived endothelial cell growth factor, and basic fibroblast growth factor promote angiogenesis and metastasis in human melanoma xenografts

Angiogenesis is a significant prognostic factor in melanoma, but the angiogenic factors controlling the neovascularization are not well defined. The purpose of this study was to investigate whether the angiogenesis and metastasis of melanoma are promoted by vascular endothelial growth factor (VEGF), interleukin 8 (IL-8), platelet-derived endothelial cell growth factor (PD-ECGF), and/or basic fibroblast growth factor (bFGF). Cells from human melanoma lines (A-07, D-12, R-18, and U-25) transplanted to BALB/c nu/nu mice were used as tumor models. Expression of angiogenic factors was studied by ELISA, Western blotting, and immunohistochemistry. Angiogenesis was assessed by using an intradermal angiogenesis assay. Lung colonization and spontaneous lung metastasis were determined after i.v. and intradermal inoculation of tumor cells, respectively. The specific roles of VEGF, IL-8, PD-ECGF, and bFGF in tumor angiogenesis, lung colonization, and spontaneous metastasis were assessed in mice treated with neutralizing antibody. The melanoma lines expressed multiple angiogenic factors, and each line showed a unique expression pattern. Multiple angiogenic factors promoted angiogenesis in the most angiogenic melanoma lines, whereas angiogenesis in the least angiogenic melanoma lines was possibly promoted solely by VEGF. Tumor growth, lung colonization, and spontaneous metastasis were controlled by the rate of angiogenesis and hence by the angiogenic factors promoting the angiogenesis. Lung colonization and spontaneous metastasis in A-07 were inhibited by treatment with neutralizing antibody against VEGF, IL-8, PD-ECGF, or bFGF. Each of these angiogenic factors may promote metastasis in melanoma, because inhibition of one of them could not be compensated for by the others. Our observations suggest that efficient antiangiogenic treatment of melanoma may require identification and blocking of common functional features of several angiogenic factors.     

Decorin suppresses tumor cell-mediated angiogenesis

The progressive growth of most neoplasms is dependent upon the establishment of new blood vessels, a process regulated by tumor-secreted factors and matrix proteins. We examined the in vitro and in vivo angiogenic ability of conditioned media obtained from fibrosarcoma, carcinoma, and osteosarcoma cells and their decorin-transfected counterparts. Human endothelial cells were investigated in vitro by evaluating three essential steps of angiogenesis: migration, attachment, and differentiation. On the whole, wild-type tumor cell-secretions enhanced endothelial cell attachment, migration, and differentiation, whereas their decorin-expressing forms inhibited these processes. Similarly, decorin-containing media suppressed endothelial cell sprouting in an ex vivo aortic ring assay. Since angiogenesis is an important component of tumor expansion, the growth rate of these cells as tumor xenografts was examined by implantation in nude mice. In vivo, the decorin-expressing tumor xenografts grew at markedly lower rates and showed a significant suppression of neovascularization. Immunohistochemical, Northern and Western blot analyses indicated that the decorin-expressing cells produced vascular endothelial growth factor (VEGF) at markedly reduced rates vis-á-vis their wild-type counterparts. Specificity of this process was confirmed by experiments where addition of recombinant decorin to the wild-type tumor cells caused 80-95% suppression of VEGF mRNA and protein. These results provide a novel mechanism of action for decorin, and indicate that decorin could adversely affect in vivo tumor growth by suppressing the endogenous tumor cell production of a powerful angiogenic stimulus.     

Hypoxia-induced angiogenesis of cultured human salivary gland carcinoma cells enhances vascular endothelial growth factor production and basic fibroblast growth factor release

The angiogenic activity of two human salivary gland tumor cell lines, ACCS from adenoid cystic carcinoma and IT-2 from mucoepidermoid carcinoma, was examined by stimulating tube formation by bovine capillary endothelial cells (BCE). ACCS and IT-2 were cultured in 20 or 3% oxygen, representing normoxic and hypoxic conditions, respectively, and conditioned medium (CM) was obtained from each culture. The BCE tubes stimulated by hypoxic CM were 1.59 (ACCS) and 1.42 (IT-2) times longer than those stimulated by normoxic CM. The tube-forming activity of CM was inhibited by preincubation with either anti-vascular endothelial growth factor (VEGF) IgG or anti-basic fibroblast growth factor (bFGF) IgG, suggesting that both VEGF and bFGF with angiogenic activity were present in the CM. This was confirmed by ELISA, which also demonstrated increased concentrations of both proteins in the hypoxic CM. Northern blot analysis showed an increased VEGF mRNA level in both carcinoma cells with hypoxia, while hypoxia did not affect the bFGF mRNA level in either cell line. The results suggest that both VEGF and bFGF are major angiogenesis factors in salivary gland tumors, and hypoxia-induced angiogenesis results from upregulation of VEGF and increased release of bFGF.     

艾迪协同卡铂高效杀伤骨肉瘤细胞的实验研究

目的：研究中药制剂艾迪对骨肉瘤化疗的增效作用及其相关机制。方法：将艾迪与卡铂单独及联合应用于骨肉瘤OS-732细胞,用MTT法检测细胞抑制率,流式细胞仪定量分析凋亡细胞所占比例,倒置相差显微镜及荧光显微镜下观察凋亡细胞形态改变,免疫细胞化学技术分析凋亡相关蛋白Fas表达。结果：艾迪与卡铂对骨肉瘤细胞都有剂量依赖性杀伤作用,艾迪浓度达到25μl/ml时,细胞抑制率为38.47%,再增加艾迪浓度抑制率不再明显改变,但如与1μg/ml卡铂合用将使细胞抑制率进一步提升为59.53%（P〈0.01）。细胞超微结构观察及凋亡率测定也显示,艾迪与卡铂联用可诱导更多骨肉瘤细胞凋亡,且联用时骨肉瘤Fas表达明显提升（P〈0.01）。结论：艾迪与小剂量卡铂合用和单用卡铂相比,可更高效杀伤骨肉瘤细胞,此作用与上调Fas表达诱导骨肉瘤细胞凋亡有关。     

The relevance of cell proliferation, vascular endothelial growth factor, and basic fibroblast growth factor production to angiogenesis and tumorigenicity in human glioma cell lines.

Tumor growth is partially dependent on angiogenesis, a process that relies on angiogenic factors. Tumorigenicity of cancer cells is thought to be associated with the production of various angiogenic factors that stimulate or inhibit the rate of endothelial cell migration and proliferation. However, the relative importance of specific individual factors originally studied in cancer cell lines has yet to be determined in vivo. In this study, we examined seven human glioma cell lines for dynamic changes of two major angiogenic factors, basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), and for doubling time and tumorigenicity in nude mice. Various correlation studies demonstrated that in these glioma cell lines, VEGF expression correlated well with RBC density in tumor sections (r2 = 0.804) and with average tumor weight (r2 = 0.987). In contrast, bFGF expression in the observed glioma cell lines did not correlate with tumorigenicity (r2 = 0.001) or with VEGF expression (r2 = 0.255). Furthermore, there was no correlation between doubling time and tumorigenicity in these cell lines (r2 = 0.160). Taken together, these results suggest that VEGF plays a major role in glioma formation and that down-regulation of VEGF, rather than bFGF, would be a more effective choice for glioma gene therapy.