Ad-Ang1-IRES-VEGF165腺病毒载体的构建及其促血管形成作用

目的:构建人血管生成素1(Ang1)和血管内皮生长因子VEGF165 (VEGF165)的共表达腺病毒载体Ad-Ang1-IRES-VEGF165(简称Ad-AV),为研究Ad-AV转基因细胞表达产物血管诱生活性提供实验依据。方法:采用IRES介导的Ang1和VEGF165双基因腺病毒共表达模式,通过常规的基因克隆和重组技术,构建Ad-AV双基因共表达腺病毒载体,经感染人胚肾QBI-293A细胞(293A)进行扩增和效价测定后,再感染WI-38人胚肺成纤维细胞,均用ELISA法检测VEGF、Ang1目的基因的表达,并采用鸡胚尿囊膜血管形成实验(CAM)法分析其对血管形成的影响。结果: 扩增的Ad-AV腺病毒效价可达4×10¹⁰pfu/ml;Ad-AV不仅能在293A细胞中成功表达目的基因Ang1、VEGF,而且在WI-38成纤维细胞也能成功表达,其表达产物具有显著的促进CAM上血管生成的活性。结论:成功构建并获得了Ad-Ang1-IRES-VEGF165重组病毒子, 目的基因均能在人胚肾和人胚肺成纤维细胞中表达,其表达产物具有诱导血管形成的功能。     

Pro-angiogenic properties of orosomucoid (ORM)

The acute phase protein orosomucoid (ORM), also known as alpha1-acid glycoprotein (AGP), is found to be increased in infection, inflammation and cancer. Recently, we demonstrated that ORM is produced by endothelial cells and detectable in urine samples of patients with bladder cancer. However, it was not clarified yet whether ORM plays a role in new vessel formation. To this aim we performed overexpression and gene silencing for ORM in human microvascular endothelial cells (HDMECs). ORM purified from human plasma was used individually or in combination with VEGF-A in endothelial tube formation, migration and proliferation assay. The in vivo effect of ORM in angiogenesis was studied using the chicken chorionallantois membrane (CAM) with subsequent counting of blood vessels on histological sections from the stimulated areas of CAM tissue. Our data show that ORM alone enhances migration but not proliferation of HDMECs. ORM alone does not induce endothelial tubes in vitro but simultaneous application of ORM with VEGF-A increases the number and the network of VEGF-A-induced endothelial tubes. Remarkably, ORM alone induces new vessel formation in vivo using CAM assay and supports the VEGF-A-induced new vessel formation in this assay. Taken together, our results let assume that ORM has pro-angiogenic properties and supports the angiogenic effect of VEGF-A. Thus, ORM seems to be involved in the regulation of angiogenesis.     

Suppression of growth and cancer-induced angiogenesis of aggressive human breast cancer cells (MDA-MB-231) on the chorioallantoic membrane of developing chicken embryos by E-peptide of pro-IGF-I

E-peptide of the pro-Insulin-like growth factor-I (pro-IGF-I) is produced from pre-pro-IGF-I by proteolytic cleavage in the post-translational processing. Previous in vitro studies conducted in our laboratory showed that Ea4-peptide of rainbow trout (rt) pro-IGF-I or Eb-peptide of human (h) pro-IGF-I exhibited activities including induction of morphological differentiation, inhibition of anchorage-independent cell growth and suppression of invasion of several well established human cancer cell lines such as MDA-MB-231, HT-29, SK-N-F1, and HepG-2 (Chen et al. [2002] Gen Comp Endocrinol 126:342-351; Kuo and Chen [2002] Exp Cell Res 280:75-89). Seeding of aggressive human breast cancer cells, MDA-MB-231, on the chorioallantoic membrane (CAM) of 5 days old chicken embryos resulted in rapid growth and invasion of the cells and induction of blood vessel formation around the MDA-MB-231 cell mass in the chicken embryos. The invasion of MDA-MB-231 cells in the chicken embryos was further confirmed by immunocytochemistry. The rapid growth and invasion of MDA-MB-231 cells and the induction of blood vessel formation by MDA-MB-231 cells on chicken CAM are inhibited by treatment with a single or multiple doses of rtEa4- or hEb-peptide. Furthermore, a dose-dependent inhibition of angiogenesis by rtEa4- or hEb-peptide was also demonstrated by the chicken CAM assay. Results of microarray analysis of human gene chips (containing 9,500 unique cDNA clones) and confirmation by comparative real-time RT-PCR analysis showed that a group of genes related to cancer cell activities are up- or down-regulated in MDA-MB-231 cells transfected with a rtEa4-peptide gene. Together these results confirm the anti-tumor activity of rtEa4- and hEb-peptides, and further suggest that these peptides could be developed as therapeutics for treating human cancers.     

The gelatin sponge-chorioallantoic membrane assay

Here we present a method for the quantification of angiogenesis and antiangiogenesis in the chick embryo chorioallantoic membrane (CAM) based on the implantation of a gelatin sponge on the top of the growing CAM on day 8 of development. After implantation, the sponge is treated with a stimulator of blood vessel formation in the absence or presence of an angiogenesis inhibitor. On day 12, blood vessels that are growing into the sponge are counted at macroscopic and microscopic levels. The estimated timeline for carrying out this protocol is 10 d. The presence of a vascular network in the CAM requires a careful analysis to distinguish new capillaries from pre-existing ones. This limitation does not occur in the avascular cornea assay, which may also take advantage of different genetic backgrounds when carried out in transgenic or knockout mice. Nevertheless, the gelatin sponge-CAM assay is simple, inexpensive and suitable for large-scale screening.     

A model system for testing gene vectors using murine tumor cells on the chorioallantoic membrane of the chick embryo

We developed a model system for testing gene vectors, based on the growth of murine tumors on the chorioallantoic membrane (CAM) of embryonic chickens. The ability of selected murine cells to grow on the CAM was rated according to the following criteria: i) formation of tumor masses; ii) metastasis formation; iii) reproducibility; iv) yield, indicated as the number of embryos surviving to assessment time with visible tumors on the CAM; v) maintainability of the cell, both in the original host and the embryonic chick, or 'shuttle maintainability'; vi) detection by the naked eye, and vii) cost/benefit relation. The murine melanoma cell lineage, B16F10, which efficiently forms distinct, pigmented tumor masses and metastases on the CAM, performed better in this model than the murine B61 cell line. In vitro transduction of B16F10 cells with a recombinant adenovirus carrying a construct of the E. coli LacZ gene followed by inoculation onto the CAM resulted in beta-galactosidase expression in the tumor mass growing on the CAM. This model is potentially applicable to preclinical evaluation of gene vectors, especially for gene therapy of cancer.     

Structure-activity relationships studies of the anti-angiogenic activities of linomide

The synthesis and anti-angiogenic activities of linomide and its analogues are reported. Three of the analogues are 3.3-69 times more potent than linomide at inhibiting blood vessel formation in the CAM angiogenesis assay. These compounds possessed considerable anti-proliferative activity against isolated HUVEC cells with no activity against epithelial-derived prostate tumor cells.     

Capillary plexus development in the day five to day six chick chorioallantoic membrane is inhibited by cytochalasin D and suramin

The chick chorioallantoic membrane (CAM) is a valuable model for evaluating angiogenesis and vasculogenesis. Our purpose was to characterize the formation of the CAM vasculature, in particular the capillary plexus, between days five and six after fertilization and to examine the mode of action of cytochalasin D and suramin on vascular development during this interval. The CAM increased 20-fold in size between days five and six, during which time the capillary plexus forms by both migration of mesodermal blood vessels toward the ectoderm and by the formation of new vessels from angioblasts near the ectoderm. Between days five and six, the CAM becomes thinner, and the density of the mesodermal cells decreases. To determine the mode of action of anti-angiogenic drugs on the day five to day six CAM, various concentrations of cytochalasin D or suramin were added directly to day five CAMs, and their effects were evaluated on day six. Both drugs significantly inhibited CAM growth, altered branching patterns of the major vessels, decreased area of the major vessels, and inhibited the formation of the capillary plexus by inhibiting both vasculogenesis and the migration of mesodermal blood vessels to the ectoderm. Cytochalasin D also inhibited compartmentalization of the plexus. Cytochalasin D and suramin were inhibitory at similar doses. This study provides new information on early CAM development, establishes the mode of action and dose dependency of cytochalasin D and suramin on day five to day six CAMs, and demonstrates that the day five to day six CAM provides a useful assay to examine the effect of anti-angiogenic drugs on blood vessel development, including capillary plexus formation.     

人血管生成素-PE40融合蛋白基因的构建、表达及活性研究

利用 PCR扩增出人血管生成素 (h ANG)成熟肽的基因片段 .与绿脓杆菌外毒素缺失突变体PE40的基因连接后 ,克隆入 p UC1 9载体中 .测序后克隆入表达载体 p RSETB,构建成 h ANG-PE40融合基因的表达载体 .IPTG诱导 ,表达出分子量约为 58k D的 His6- ANG- PE40融合蛋白 ,占菌体总蛋白的 8% .Ni2 +- NTA树脂纯化表达蛋白 ,SDS- PAGE结果显示纯化重组蛋白为单一条带 .鸡胚绒毛尿囊膜鉴定表明重组蛋白体外能够有效地抑制血管的形成 .     

人血管生成素cDNA的克隆与表达

血管生成素 ( angiogenin,ANG)广泛存在于多种肿瘤组织中 ,在肿瘤发生的不同阶段刺激新生血管的形成 .利用 RT- PCR方法从培养的人肺癌细胞系 A549扩增得到了 ANG c DNA片段 ,测序正确后克隆入融合表达载体 p RSETB中 ,构建了原核表达菌株 .经 IPTG诱导 ,表达了 N端融合His6的 ANG融合蛋白 ,表达量占菌体总蛋白的 1 0 % ,纯化后的血管生成素体外能够有效地刺激鸡胚绒毛尿囊膜的血管形成 .     

Collagenolysis-dependent angiogenesis mediated by matrix metalloproteinase-13 (collagenase-3)

We have demonstrated previously that new blood vessel formation induced by angiogenic growth factors in onplants placed on the chorioallantoic membrane (CAM) of the chick embryos is critically dependent on the cleavage of fibrillar collagen by a previously unidentified interstitial collagenase. In the present study we have used a quantitative CAM angiogenesis system to search for and functionally characterize host avian collagenases responsible for the collagen remodeling associated with angiogenesis. Among the matrix metalloproteinases (MMPs) identified in the CAM onplant tissue, the chicken MMP-13 (chMMP-13) was the only enzyme whose induction and expression coincided with the onset of angiogenesis and blood vessel formation. The chMMP-13 cDNA has been cloned and recombinantly expressed. The chMMP-13 protein has been purified, characterized in vitro, and examined in situ in the CAM. MMP-13-positive cells appear in the CAM shortly after angiogenic stimulation and then accumulate in the collagen onplant tissue. Morphologically, the chMMP-13-containing cells appear as hematopoietic cells of monocyte/macrophage lineage. In vitro, the chMMP-13 proenzyme is rapidly and efficiently activated through the urokinase plasminogen activator/plasminogen/plasmin cascade into a collagenase capable of cleaving native but not the (r/r) mutant collagenase-resistant collagen. Surprisingly, nanogram levels of purified chMMP-13 elicit an angiogenic response in the CAM onplants comparable with that induced by the angiogenic growth factors. The chMMP-13-mediated response was efficiently blocked by select protease inhibitors indicating that plasmin-activated chMMP-13 can function as an angiogenic factor in vivo. Altogether, the results of this study extend the physiological role of MMP-13, previously associated with cartilage/bone resorption, to the collagen remodeling involved in the angiogenic cascade.     

Hepatoma-derived growth factor is a pulmonary endothelial cell-expressed angiogenic factor

Hepatoma-derived growth factor (HDGF) was previously identified as a developmentally regulated cardiovascular and renal gene that is mitogenic for vascular smooth muscle and aortic endothelial cells. As reciprocal interactions of smooth muscle and endothelial cells are necessary for vascular formation, we examined whether HDGF plays a role in angiogenesis. According to immunohistochemistry, HDGF was highly expressed in endothelial cells of nonmuscularized, forming blood vessels of the fetal lung. HDGF was also expressed in endothelial cells of small (20 microm) mature arteries and veins. By Western immunoblotting, HDGF was highly expressed by human pulmonary microvascular endothelial cells in vitro. Adenoviral overexpression of HDGF was mitogenic for human pulmonary microvascular endothelial cells in serum-free medium, stimulating a 1.75-fold increase in bromodeoxyuridine (BrdU) uptake and a twofold increase in cell migration. With the chick chorioallantoic membrane (CAM), a biologic assay for angiogenesis, exogenous recombinant HDGF significantly stimulated blood vessel formation and a dose-dependent reorganization of cells within the CAM into a more compact, linear alignment reminiscent of tube formation. According to double immunostaining for endothelial cells with a transforming growth factor-betaII receptor antibody and BrdU as a marker of cell proliferation, exogenous HDGF selectively stimulated endothelial cell BrdU uptake. HDGF also activated specific ERK1/2 signaling and did not overlap with VEGF SAPK/JNK, Akt-mediated pathways. We conclude that HDGF is a highly expressed vascular endothelial cell protein in vivo and is a potent endothelial mitogen and regulator of endothelial cell migration by mechanisms distinct from VEGF.     

The hepatitis C virus internal ribosome entry site facilitates efficient protein synthesis in blood vessel endothelium during tumour angiogenesis

The development of gene delivery systems for therapeutic use involves vectors (often retrovirus or adenovirus) which typically encode one target protein, but the use of internal ribosome entry sites (IRES) can confer the ability to express more than one protein from bi- or polycistronic mRNAs. IRES elements can display tissue-specific expression, so it is necessary to determine suitable IRES for specific clinical applicability. Blood vessel endothelial cells are important clinically since many different conditions involve neo-vascularisation (angiogenesis). We have demonstrated that the viral hepatitis C IRES element is a powerful mediator of protein synthesis in angiogenesis, such as found in solid tumours. Homologous recombination was used to introduce IRES-lacZ sequences into the Lmo2 gene, which is expressed in endothelial cells. β-Galactosidase expression was determined during vascular remodelling in mouse embryos and in sprouting endothelium during growth of solid tumours, and showed that the hepatitis C IRES is used efficiently for protein synthesis in endothelial cells. This IRES element can provide the means to express two or more therapeutic genes in blood vessel endothelium in clinical conditions, such as cancer, which depend on angiogenesis.     

内皮细胞粘附分子与血管壁通透性

内皮细胞是血管壁的主要通透屏障,对血管壁通透性有多种调节作用。细胞粘附分参与细胞间连接形成以及细胞－基底膜间的粘附,与内皮层连续完整性以及通透性密切相关。参与内皮细胞中间连接的粘附分子有三大家庭,即整合素家庭、钙依赖性粘附素家族和免疫球蛋白家族,对这些粘附分子家族的研究有助于阐明内皮细胞损伤修复、新生血管形成、血管通透性调节以及循环血细胞游出的机制。     

健脾化瘀中药提高胞嘧啶脱氨酶/单纯疱疹病毒胸苷激酶基因治疗肝细胞癌的实验研究

目的:观察健脾化瘀中药提高胞嘧啶脱氨酶/单纯疱疹病毒胸苷激酶基因治疗肝细胞癌的作用。方法:脂质体lipofectamine将含有双自杀基因的腺病毒载体pAd-CD/TK导人293细胞,收集病毒上清转染人肝癌细胞BEL7402,MTT法测定BEL7402细胞存活率。裸鼠人肝癌模型转染CD/TK双自杀基因后,给予5-FC500mg/kg,GCV 100mg/kg腹腔注射,同时予健脾化瘀中药960复方灌胃。观察肿瘤生长情况。结果:给予前体药物5-FC和GCV后,CD/TK转染细胞被杀死。并表现出较强的旁观者效应。转染细胞比例达到10%即表现出较强的杀伤作用(P<0.01)。健脾化瘀中药960复方具有提高旁观者效应作用,1.67ml/kg和2.5ml/kg960复方含药血清组细胞存活率显著低于对照组(P<0.01)。转染基因组应用5-FC和GCV治疗后,裸鼠肝癌的生长明显受到抑制(P<0.05),抑瘤率39.42%,单用中药组抑瘤率18.04%,中药与CD/TK 5-FC/GCV联合运用组,较单纯CD/5-FC/HSV-tk/GCV对裸鼠肿瘤模型的生长抑制作用更加明显(P<0.05),抑瘤率55.10%。结伦:腺病毒介导CD/TK自杀基因可有效地杀死人肝癌BEL7402细胞,健脾化瘀中药960复方具有显著提高CD/TK双自杀基因对人肝癌细胞的抑杀作用。     

Dexamethasone interferes with osteoblasts formation during osteogenesis through altering IGF-1-mediated angiogenesis

Dexamethasone (Dex), a synthetic glucocorticoid (GC) with long-lasting treatment effects, has been proved to exert a modulatory effect on osteoblast proliferation and differentiation during embryonic osteogenesis. However, it is still controversial if Dex exposure influences endochondral ossification and the underlying mechanism. In this study, chick embryos in vivo and preosteoblast cell cultures in vitro were utilized to investigate the effects of Dex on osteoblast formation and differentiation during the skeletal development. We first demonstrated that Dex exposure could shorten the long bones of 17-day chick embryos in vivo, and also downregulated the expressions of osteogenesis-related genes. Next, we established that Dex exposure inhibited the proliferation and viability of preosteoblasts-MC3TC-E1 cells, and the addition of insulin-like growth factor 1 (IGF-1) could dramatically rescue these negative effects. On the basis of remarkable changes in the rescue experiments, we next verified the important role of angiogenesis in osteogenesis by culturing isolated embryonic phalanges in Dulbecco's modified Eagle's medium culture or on the chick chorioallantoic membrane (CAM). Then, we transplanted MC3T3-E1 cell masses onto the CAM. The data showed that Dex exposure reduced the vessel density within the developed cell mass, concomitantly with the downregulation of IGF-1 pathway. We verified that the inhibition of blood vessel formation caused by Dex could be rescued by IGF-1 treatment using the CAM angiogenesis model. Eventually, we demonstrated that the shortened length of the phalanges in the presence of Dex could be reversed by IGF-1 addition. In summary, these findings suggested that the inhibition of Igf-1 signal caused by Dex exposure exerts a detrimental impact on the formation of osteoblasts and angiogenesis, which consequently shortens long bones during osteogenesis.     

Immobilization of aprotinin to fibrinogen as a novel method for controlling degradation of fibrin gels

The goal of this work was to demonstrate that aprotinin conjugated to fibrinogen could (1) maintain its function and (2) control fibrin degradation. Using the chick chorioallantoic membrane (CAM) assay, we found that blood vessels did not directly invade fibrin constructs containing immobilized fibroblast growth factor-2. Because the fibrin quickly degraded within approximately 5 days, we hypothesized that controlling fibrinolysis may improve direct blood vessel invasion. Aprotinin, a protease inhibitor typically added to slow fibrinolysis, is a small protein and can diffuse out of the gel resulting in the loss of fibrinolysis protection. Therefore, using a novel synthesis strategy, aprotinin and a fluorescent reporter, Cy3, were chemically conjugated to fibrinogen. In vitro microplate absorbance assays showed that the conjugated aprotinin was able to inhibit plasmin-mediated fibrin degradation and that its activity was comparable to equimolar levels of soluble, nonconjugated aprotinin. Additionally, we found that fibrinolysis rates could be tuned by varying the level of conjugated aprotinin within the gel. The conjugated aprotinin also demonstrated functionality in vivo. In the chick CAM assay, fibrin gels containing conjugated aprotinin were approximately 5 times larger than gels containing soluble aprotinin after 4 days. Also, in support of our hypothesis, we found that immobilized aprotinin within fibrin gels demonstrated substantial blood vessel invasion.     

Effect of green tea polyphenols on angiogenesis induced by an angiogenin-like protein

Angiogenesis is a fundamental process by which new blood vessels are formed. The angiogenesis process is induced by several growth factors. Among them angiogenin is the most potent blood vessel inducer known. In this paper, we have investigated the effect of green tea polyphenols, mainly the catechins, on an angiogenin-like protein induced angiogenesis process. The angiogenin-like protein was isolated from goat serum and the effect of green tea components was tested by the chicken chorioallantoic membrane (CAM) assay. The results show that green tea components are capable of reducing the vascularization on CAM that is induced by the angiogenin-like protein.     

Mitogenic activity of chicken chorioallantoic fluid is temporally correlated to vascular growth in the chorioallantoic membrane and related to fibroblast growth factors

The chorioallantoic membrane (CAM) is one of the most vascularized tissues in the chicken embryo. Capillary growth proceeds until day 10 of development and thereafter abruptly regresses. As it is generally accepted that the formation of new blood vessel is regulated by growth factors, we have investigated the presence of angiogenic and mitogenic factors in the chicken chorioallantois. In the present study, we show that chorioallantoic fluid (CAF) contains angiogenic substances that are probably synthesized in the CAM or the embryonic kidney. When applied in the chorioallantoic membrane assay, CAF from 9 day chicken embryos elicits a strong angiogenic response. This angiogenic activity of CAF is associated with pronounced mitogenic effects in vitro. Comparison of different embryonic fluids reveals that mitogenic activity is particularly evident in the CAF but not detectable in embryonic serum and amnion fluid. Expression of mitogenic activity is found to be temporally correlated with vascular growth in the CAM. High activity is detected in CAF prior to day 10 and then sharply decreases, thus preceding termination of capillary growth by one day. Heparin-sepharose affinity chromatography suggests that the biological activities of CAF probably correspond to the presence of acidic and basic fibroblast growth factor (aFGF and bFGF). In Western blot analyses of CAF, an immunoreactive bFGF-like protein of about 17 x 10(3) Mr is recognized by a monospecific anti-bFGF antiserum. This protein elutes at 2.4 M NaCl from the heparin-sepharose. The mitogenic activity of the CAF can be specifically blocked by the anti-bFGF antibody indicating bFGF to be the active mitogenic principle of the CAF. These results strongly suggest that basic and probably acidic FGF play an important role in the regulation of chorioallantoic vascular growth.