共价-离子结合肝素改性对牛心包膜抗凝血性能的影响

目的在碳化二亚胺交联的去细胞牛心包膜上采用3种方式进行肝素改性,即共价键合肝素、离子结合肝素以及共价-离子联合负载肝素,比较这3种肝素化方式对牛心包膜抗凝血性能的影响,并筛选出最佳肝素化方式。方法通过溶血试验、血小板黏附实验、体外凝血测试以及复钙时间测定观察3种方式肝素化的基质抗凝血性能和血栓形成情况,以评价其血液相容性。结果综合上述4项检测的结果,共价-离子联合负载肝素改性的抗凝血性能优于共价键合肝素、离子结合肝素这2种单独负载肝素方式,溶血率〈5％,电镜照片无血小板黏附,体外生理盐水浸泡15d后,仍保持良好的抗凝血活性。结论共价-离子联合负载肝素,将离子结合肝素活性好、共价键合肝素稳定性强的优点结合起来,取长补短,使这种方式肝素改性的牛心包具有良好的血液相容性。     

Modulation of angiogenic potential of collagen matrices by covalent incorporation of heparin and loading with vascular endothelial growth factor

One of the prominent shortcomings of matrices for tissue engineering is their poor ability to support angiogenesis. We report here on experiments to enhance the angiogenic properties of collagen matrices. Our aim is to achieve this goal by covalently incorporating heparin into collagen matrices and by physically immobilizing angiogenic vascular endothelial growth factor (VEGF) to the heparin. The immobilization of heparin was performed with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS). Carboxyl groups on the heparin are activated to succinimidyl esters, which react with amino functions on the collagen to zero length cross-links. This modification leads--in addition to the incorporation of heparin--to gross changes in in vitro degradation behavior and water-binding capacity. As a first approach to testing angiogenic capabilities, endothelial cells were exposed to nonmodified and heparinized collagen matrices. This exposure leads to an increase in endothelial cell proliferation. The increase can be further enhanced by loading the (heparinized) collagen matrices with VEGF. Evaluation of the angiogenic potential of heparinized matrices was further investigated by exposing them to the chorioallantoic membrane of chicken embryos and to the subcutaneous tissue of rats. Both approaches show that heparinized matrices have substantially increased angiogenic potential. In particular, the loading of heparinized matrices with VEGF invokes a further increase in angiogenic potential. It is apparent that the physical binding of VEGF to heparin allows for a release that is beneficial to angiogenesis. By varying the heparin and EDC/NHS concentrations during the modification process and by varying the loading with VEGF, the angiogenic potential as well as the degradation behavior can be adapted to obtain matrices that fulfill specific angiogenic requirements in the field of tissue engineering.     

Influence of modified extracellular matrices on TI6AL4V implants on binding and release of VEGF

Besides osteoconductive and osteoinductive signals, angiogenesis plays a crucial role in bone development and regeneration and consequently in the integration of implants. Therefore we investigated in this study the binding and release behaviour of vascular endothelial growth factor (VEGF) from Ti6Al4V surfaces coated with 3-dimensional collageneous matrices, some additionally modified with heparin. The heparin was incorporated using different methods: a) adsorptive immobilization b) crosslinking with N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) or c) incorporating during self-assembly of fibrils followed by cross-linking. For unmodified samples, maximum VEGF adsorption was reached with 85 ng VEGF/cm(2). On all 3d-collagen coated surfaces studied (with or without heparin), no saturation could be observed in the range of 0-256 ng VEGF/cm(2).Improved release kinetics were observed for the modified coatings. The initial burst of VEGF within the first 24 h was diminished. From the third day of delivery heparinized matrices showed a higher release of VEGF than the pure collagen matrix and the unmodified reference surface, respectively. In vitro, the proliferation of human dermal microvascular endothelial cells was increased with released VEGF from all investigated samples compared to a VEGF-free control. After 7 days highest increases in cell numbers were observed with solutions from heparinized matrices.It is concluded that functionalization of Ti6Al4V surfaces with heparinized collageneous matrices and VEGF leads to advantageous properties concerning the impact on angiogenesis and thus may improve bone regeneration in the microenvironment of implants.     

肝素化羊脱细胞真皮基质对深Ⅱ度烧伤创面组织中血管内皮生长因子和层粘连蛋白的影响

目的研究肝素化羊脱细胞真皮基质(ADM)促进深Ⅱ度烧伤创面愈合的作用机制。 方法制作大鼠深Ⅱ度烧伤模型,随机分为碘伏纱布组、猪ADM组、羊ADM组和肝素化羊ADM组,每组40只。在伤后不同时间段,观察各组创面愈合率,同时在各个时间段处死大鼠8只,取创面组织标本做RT-qPCR和蛋白质印迹法检测各组创面组织中血管内皮生长因子(VEGF)和层粘连蛋白(LN)的mRNA与蛋白表达情况。对数据行单因素方差分析和t检验。 结果肝素化羊ADM组深Ⅱ度创面的愈合率优于碘伏纱布组。RT-qPCR检测发现,VEGF的mRNA在各组伤后3 d的创面组织中表达较对照组高,各组间的比较发现,伤后7 d,肝素化羊ADM组的VEGF的mRNA表达量明显高于碘伏纱布组(t＝80.83,P<0.05),羊ADM组和猪ADM组在伤后7 d也较碘伏纱布组的表达量高(t＝60.80、53.42,P值均小于0.05);蛋白质印迹法检测肝素化羊ADM组伤后3 d时VEGF蛋白表达量较多,随后便逐渐减少,伤后21 d时蛋白表达较少。伤后3 d,碘伏纱布组LN在创面的表达明显下降,在之后的治疗过程中,LN的表达逐渐恢复,伤后14 d的表达量明显高于伤后3 d(t＝19.5,P<0.001),伤后21、28 d的表达量与伤后14 d相差不明显。蛋白质印迹法检测发现肝素化羊ADM组LN在伤后3 d便开始出现表达量逐渐增多,伤后21 d时出现蛋白表达较多。 结论肝素化羊ADM能够保护创面及促进创面组织中VEGF和LN的表达,进而促进创面愈合。     

Effects of vascular endothelial growth factor released from cultured dermal substitute on proliferation of vascular endothelial cells in vitro

Allogeneic cultured dermal substitute (CDS) was prepared by culturing fibroblasts on a two-layered spongy matrix of hyaluronic acid and atelocollagen. CDS can be cryopreserved and transported to other hospitals in a frozen state. The present study was designed to analyze the amounts of vascular endothelial growth factor (VEGF) released from fibroblasts in fresh and cryopreserved CDS and to investigate the effects of this VEGF on proliferation of vascular endothelial cells in vitro. The culture medium used in preparing CDS (fresh CDS culture medium sample) was collected and stored at –30°C for the quantitative analysis of VEGF. After thawing cryopreserved CDS, it was recultured in a culture medium for 1 week. The culture medium used was collected and stored at –30°C for quantitative analysis of VEGF. The amounts of VEGF released from the fresh and cryopreserved CDS into the culture medium were about 610pg/ml and 640pg/ml, respectively. This finding suggests that the cryopreserved CDS retains its ability to release VEGF. Immunohistological analysis indicated that some of the VEGF adhered to the matrix. Human vascular endothelial cells were cultured in medium mixed with the fresh or cryopreserved CDS culture medium sample. Proliferation of vascular endothelial cells was enhanced by increasing the concentration of both CDS culture medium samples. When antihuman VEGF antibody was added to the culture medium, the proliferative activity of vascular endothelial cells was reduced. These findings confirm that VEGF released from CDS promotes proliferation of vascular endothelial cells.     

Effects of modified collagen matrices on human umbilical vein endothelial cells

The most commonly used biomaterials fail to ensure sufficient angiogenesis for fast in vivo incorporation. This results in central necrosis and consequent infection. One way of obtaining a high angiogenic response is the application of vascular endothelial growth factor (VEGF). To obtain a sustained release of these cytokines, heparin was incorporated into collagen matrices using 1-ethyl-3(3-dimethyl-aminopropyl) carbodiimide (EDC) and N-hydroxysuccinmide (NHS). The functionality of the heparinized collagen matrices was then enhanced by immobilization of VEGF via its heparin-binding domain. This procedure changed in vitro degradation behavior and water-binding capacity. Accelerated endothelial cell proliferation was also achieved. A range of different heparin and EDC/NHS concentrations in combination with VEGF induced variation in endothelial cell growth and tubulogenic formation. Polymerized collagen scaffolds presented biointeractive systems with integrated angiogenic activity. This may become a useful tool in the clinical therapy of disorders connected with wound healing.     

Gradient nanofibrous chitosan/poly ɛ-caprolactone scaffolds as extracellular microenvironments for vascular tissue engineering

One of the major challenges of tissue-engineered small-diameter blood vessels is restenosis caused by thrombopoiesis. The goal of this study was to develop a 3D gradient heparinized nanofibrous scaffold, aiding endothelial cells lined on the lumen of blood vessel to prevent thrombosis. The vertical graded chitosan/poly ?-caprolactone (CS/PCL) nanofibrous vessel scaffolds were fabricated with chitosan and PCL by sequential quantity grading co-electrospinning. To mimic the natural blood vessel microenvironment, we used heparinization and immobilization of vascular endothelial growth factor (VEGF) in the gradient CS/PCL. The quantity of heparinized chitosan nanofibers increased gradually from the tunica adventitia to the lumen surfaces in the gradient CS/PCL wall of tissue engineered vessel. More heparin reacted to chitosan nanofiber in gradient CS/PCL than in uniform CS/PCL nanofibrous scaffolds. Antithrombogenic properties of the scaffolds were enhanced by the heparinization of these scaffolds, as shown by activated partial thromboplastin time and platelet adhesion assay. Compared to the uniform CS/PCL scaffold, the release of VEGF from the gradient CS/PCL was more stable and sustained, and the burst release of VEGF was reduced approximately 42.5% within the initial 12 h. The adhesion and proliferation of human umbilical vein endothelial cells (HUVEC) were enhanced on the gradient CS/PCL scaffold. Furthermore, HUVEC grew and formed an entire monolayer on the top side of the gradient CS/PCL scaffold. Therefore, use of vertical gradient heparinized CS/PCL nanofibrous scaffolds could provide an approach to create small-diameter blood vessel grafts with innate properties of mammalian vessels of anticoagulation and rapid induction of re-endothelialization.     

休克淋巴液对大鼠内皮细胞VEGF表达的影响

目的观察休克淋巴液对大鼠肺微血管内皮细胞(PMVEC)、肠系膜微淋巴管内皮细胞(MMLEC)、血管内皮生长因子(VEGF)表达的影响。方法无菌条件下复制大鼠重症失血性休克模型,引流休克时肠系膜淋巴液或收集门静脉血,同时收集正常淋巴液及门静脉血作为对照。以4%终浓度的处理因素与第3代PMVEC及MMLEC共同孵育6h,RT-PCR测定VEGF mRNA表达。结果不同处理因素与PMVEC及MMLEC孵育6h后,休克淋巴液组两种内皮细胞的VEGF mRNA表达均显著低于休克血浆组、正常淋巴液组、正常血浆组、胎牛血清(FBS)组、DMEM组;休克血浆组显著低于正常淋巴液组、正常血浆组、FBS组和DMEM组;其它组间无统计学差异。结论休克淋巴液可抑制内皮细胞的VEGF mRNA表达,且作用强于休克血浆。     

Binding and release of basic fibroblast growth factor from heparinized collagen matrices.

Endothelial cell seeding is a promising method to improve the performance of small-diameter vascular grafts. Growth of endothelial cells seeded on the luminal surface of synthetic vascular grafts, coated with a matrix suitable for cell seeding (e.g. collagen), can be accelerated by local, sustained release of basic fibroblast growth factor (bFGF). In this study two potential matrices for in vivo endothelial cell seeding were studied with respect to bFGF binding and release: collagen crosslinked using N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS), as well as heparinized EDC/NHS-crosslinked collagen. bFGF binding was determined after incubation of circular samples (10 mm diameter) with 0.25 ml bFGF solution for 90 min. Immobilization of increasing amounts of heparin, also using EDC and NHS, to crosslinked collagen containing 14 free primary amino groups per 1000 amino acid residues (E/N14C) resulted in binding of increasing amounts of bFGF. A plateau in bFGF binding was observed for heparinized E/N14C containing approximately 2.0-3.0 wt% of immobilized heparin which was obtained using a molar ratio of EDC to heparin-carboxylic acid groups of 0.4 during heparin immobilization (E/N14C-H(0.4)). At concentrations up to 840 ng bFGF/ml, 10% of the added bFGF bound to E/N14C, while binding of bFGF to E/N14C-H(0.4) amounted to 22%. Both E/N14C and E/N14C-H(0.4) pre-loaded with bFGF showed sustained bFGF release. A burst release of 30% in endothelial cell culture medium (CM) was observed for E/N14C during the first 6 h, compared to 2% release from E/N14C-H(0.4). After 28 days, the bFGF release from E/N14C and E/N14C-H(0.4) in CM amounted to 100 and 65%, respectively. Combined results of binding and release of bFGF indicate that compared to E/N14C, E/N14C-H(0.4) is the substrate of choice for bFGF pre-loading and subsequent endothelial cell seeding.     

Acellular biological tissues containing inherent glycosaminoglycans for loading basic fibroblast growth factor promote angiogenesis and tissue regeneration

It was found in our previous study that acellular tissues derived from bovine pericardia consist primarily of insoluble collagen, elastin, and tightly bound glycosaminoglycans (GAGs). It is speculated that the inherent GAGs in acellular tissues may serve as a reservoir for loading basic fibroblast growth factor (bFGF) and promote angiogenesis and tissue regeneration. This study was therefore designed to investigate effects of the content of GAGs in acellular bovine pericardia on the binding of bFGF and its release profile in vitro while its stimulation in angiogenesis and tissue regeneration in vivo were evaluated subcutaneously in a rat model. To control the content of GAGs, acellular tissues were treated additionally with hyaluronidase for 1 (Hase-D1), 3 (Hase-D3), or 5 days (Hase-D5). The in vitro results indicated that a higher content of GAGs in the acellular tissue resulted in an increase in bFGF binding and in a more gradual and sustained release of the growth factor. The in vivo results obtained at 1 week postoperatively showed that the density and the depth of neo-vessels infiltrated into the acellular tissue loaded with bFGF (acellular/bFGF) were significantly greater than the other test samples. At 1 month postoperatively, vascularized neo-connective tissues were found to fill the pores within each test sample, particularly for the acellular/bFGF tissue. These results suggested that the sustained release of bFGF from the acellular/ bFGF tissue continued to be effective in enhancing angiogenesis and generation of new tissues. In conclusion, the inherent GAGs present in acellular tissues may be used for binding and sustained release of bFGF to enhance angiogenesis and tissue regeneration.     

Papillary carcinoma of the thyroid: evidence for a role for hepatocyte growth factor (HGF) in promoting tumour angiogenesis

The pattern of vascularization of papillary carcinoma was investigated in tumour sections from 31 cases and in primary cultures from 12 cases. Tumour sections were immunostained for von Willebrand Factor (vWF) to visualize blood vessels; for endothelial-specific nitric-oxide-synthase (EC-NOS), as a marker of endothelial cell activation; and for Ki-67 to evaluate endothelial cell proliferation. It was found that endothelial cells lining venous vessels located in peritumoural fibrous tissue were intensely EC-NOS-positive and occasionally Ki-67-positive. Capillary vessels of tumour papillae were not stained for Ki-67 and were weakly EC-NOS-positive. Primary cultures of papillary carcinoma cells were used as a potential source of factors active on endothelial cells. It was found that thyroid tumour cells contain RNAs for angiopoietin, vascular endothelial growth factor (VEGF), and VEGF-C; moreover, they release large amounts of VEGF into culture supernatants and exert chemotactic activity in vitro for the endothelial cell line SIEC. The ability of papillary carcinoma cells to release angiogenic factors could be stimulated in vitro. Hepatocyte growth factor (HGF; 25 ng/ml) induced a 1.2- to 5-fold increase in the amount of VEGF released by tumour cells and a 1.2- to 4.2-fold increase in the amount of chemotactic activity present in culture supernatants. Met protein, the high affinity HGF-receptor, is overexpressed in a large proportion of cases of papillary carcinoma. These findings are consistent with the possibility that HGF-Met protein interaction is one of the molecular mechanisms promoting the vascularization of papillary carcinoma of the thyroid.     

Heparinized PCL/keratin mats for vascular tissue engineering scaffold with potential of catalytic nitric oxide generation

Abstract

Heparins are capable of improving blood compatibility, enhancing HUVEC viability, while inhibiting HUASMC proliferation. Combination of biodegradable poly(ε-caprolactone) (PCL) with keratin and heparins would provide an anticoagulant and endothelialization supporting environment for vascular tissue engineering. Herein, PCL and keratin were first coelectrospun and then covalently conjugated with heparins. The resulting mats were surface-characterized by ATR-FTIR, SEM, WCA, and XPS. Cell viability data showed that the heparinized PCL/keratin mats could motivate the adhesion and growth of HUVEC, while inhibit HUASMC proliferation. In addition, these mats could prolong blood clotting time and reduce platelet adhesion as well as no erythrolysis. Interestingly, these mats could catalyze the NO donor in blood to release NO, which could enhance endothelial cell growth, while decrease smooth muscle cell proliferation and platelet adhesion. In summary, the heparinized mats would be a good candidate as a scaffold for vascular tissue engineering. This study is novel in that we prepared a type of heparinized tissue scaffold that could catalyze the NO donor to release NO to regulate endothelialization without angiogenesis and thrombus formation.     

Characterization of a cultured dermal substitute composed of a spongy matrix of hyaluronic acid and collagen combined with fibroblasts

 The authors have developed an allogeneic cultured dermal substitute (CDS) through cultivation of fibroblasts on a two-layered spongy matrix of hyaluronic acid (HA) and atelocollagen (Col). The Col spongy layer is essential for attachment and proliferation of fibroblasts on the two-layered spongy matrix. The HA spongy layer is necessary for maintaining the moisture environment on the wound surface. The optimal weight ratio of HA/Col is determined by considering the following characteristics: mechanical properties for handling, cell viability after thawing, potency of vascular endothelial growth factor (VEGF) release after thawing, efficacy of wound healing, and manufacturing cost. This study is designed to investigate the physical properties for handling, the growth behavior of fibroblasts on the spongy matrix, and the quantitative analysis of VEGF released from fibroblasts in the fresh or cryopreserved CDS. The results of this study suggest that a CDS composed of Col spongy matrix alone has the highest potency in regard to the release of VEGF. However, taking into account the manufacturing cost, coupled with the potency of VEGF release, a two-layered sponge of HA and Col with a weight ratio of 5/2 is very promising for commercial application. Received: October 4, 2002 / Accepted: March 20, 2003     

人血管内皮生长因子基因的克隆、表达及生物活性分析

目的 克隆人血管内皮细胞生长因子165(VEGF165)基因，构建真核表达载体，观察其对脐静脉内皮细胞的增殖作用和血管新生的影响。方法 利用RT-PCR方法，从人扁桃体组织中扩增人VEGF165 cDNA完整编码区，并构建成pcDNA3．1( )／VEGF165(简称pcDNA／V)重组体；应用脂质体介导的基因转移技术将构建的真核表达载体pcDNA／V体外转染至人脐静脉内皮细胞(HUVEC)，MTT法检测其对内皮细胞增殖的影响。建立家兔下肢缺血模型，注射重组质粒pcDNA／V，pcDNA3．1( )空质粒作对照，选取不同时间点，行血管造影。结果 构建的真核表达载体pcDNA／V的酶切电泳分析和测序表明结果正确。pcDNA／V转染HUVEC能明显促进内皮细胞的分裂增殖。血管造影显示，术后基因治疗组远端动脉充盈早于对照组，新生血管数目也明显多于同时期对照组。结论 成功克隆了人VEGF165基因，构建了其真核表达载体。体内外生物学活性研究证实，重组质粒的表达产物具有刺激HUVEC增殖和促进缺血肢体侧枝循环建立的功能。     

Modification of collagen matrices for enhancing angiogenesis

The vascularization of engineered tissues in many cases does not keep up with the ingrowth of cells. Nutrient and oxygen supply are not sufficient, which ultimately leads to the death of the invading cells. The enhancement of the angiogenic capabilities of engineered tissues therefore represents a major challenge in the field of tissue engineering. The immobilization of angiogenic growth factors may be useful for enhancing angiogenesis. The most potent angiogenic growth factor specific to endothelial cells, vascular endothelial growth factor (VEGF), occurs in several splice variants. The variant with 165 amino acids both has a high angiogenic activity and a high affinity for heparin. We therefore incorporated heparin molecules into collagen matrices by covalently cross-linking them to amino functions on the collagen. Physical binding of VEGF to the heparin may then prevent a rapid clearance from the implant, while the release rate may become coupled to the degradation of the collagen matrix. The modified matrices were characterized by determination of the extent of the heparin immobilization, the in vitro degradation rate by collagenase. For testing the angiogenic properties, non-modified and heparinized collagen specimens were--either loaded with VEGF or non-loaded--subcutaneously implanted on the back of rats. Specimens were explanted after varying periods of implantation, the dry weights and the hemoglobin contents, as well as immunostained histological sections were evaluated: heparinized collagen matrices loaded with VEGF are vascularized to a substantially higher extent as compared to non-modified matrices.     

硫修饰脂质体包裹VEGF反义寡核苷酸对肺癌血管生成及转移的抑制作用

目的：探讨硫修饰脂质体包裹的VEGF反义寡核苷酸对肺癌血管生成和转移的抑制作用。方法：将硫修饰脂质体包裹的VEGF反义寡核苷酸（ASODN）、正义寡核苷酸（SODN）、错义寡核苷酸（MODN）加入培养的Lewis肺癌细胞中，采用免疫组织化学方法检测肺癌细胞中VEGF蛋白表达，观察经ODN处理的条件培养基对牛主动脉内皮细胞增殖的影响。另外，复制小鼠Lems肺癌模型40只，随机分为对照组、VEGFASODN组、VEGFSODN组及VEGFMSODN组，每组10只，接种肿瘤细胞24小时内，给予脂质体、VEGFASODN、VEGFSODN、VEGFMSODN皮下注射，每周2次，连续4周。检测皮下肿瘤的变化和肺转移率，并用免疫组织化学方法检测肿瘤组织微血管密度（MVD），超声检测肿瘤组织Ps、砌变化。结果：ASODN能够下调肺癌细胞中VEGF蛋白的表达，经ASODN处理的条件培养基能显著抑制牛主动脉内皮细胞的增殖。ASODN组小鼠肿瘤生长及肺转移受到显著抑制，MVD、PS、RI与其它各组相比有明显差异（P〈0．01）。结论：硫修饰脂质体包裹的VEGF反义寡核苷酸能够显著抑制肺癌血管生成，进而抑制肿瘤生长及转移。     

PEG-cross-linked heparin is an affinity hydrogel for sustained release of vascular endothelial growth factor

An affinity-based controlled release system for growth factors having heparin-binding domains was prepared using a cross-linked heparin gel. The heparin gel was made by reacting hydrazide-functionalized heparin (Hep-ADH) with the N-hydroxysuccinimidyl ester of poly(ethylene glycol)-bis-butanoic acid (SBA-PEG-SBA). The degree of cross-linking could be controlled by defining the stoichiometry of hydrazide modification and the PEG cross-linker addition. The release of vascular endothelial growth factor (VEGF) was characterized as a heparin-binding growth factor. VEGF was directly injected into the heparin gel and the loaded VEGF displayed a slow, controlled release over 3 weeks with little initial burst phase. The biological activity of the released VEGF was measured with a proliferation assay utilizing human umbilical vein endothelial cells. The released VEGF maintained its biological activity at all time points investigated. The heparin gel with loaded VEGF was implanted sub-cutaneously in the dorsal region of mice. A significantly increased density of the endothelial cell marker platelet endothelial adhesion molecule (PECAM-1) was observed in histological specimens of the tissues surrounding the implanted gel.     

当归补血汤对动脉粥样硬化兔内皮祖细胞及血清VEGF、SDF-1的影响

目的: 观察当归补血汤对兔动脉粥样硬化模型外周血内皮祖细胞(endothelial progenitor cells, EPCs)功能及血清血管内皮生长因子(vascular endothelial growth factor, VEGF)、基质细胞衍生因子1(stromal cell-derived factor 1,SDF-1)表达的影响。方法: 新西兰兔25只,免疫损伤结合高脂饮食法建立动脉粥样硬化模型,模型动物随机分为5组,每组5只。模型组灌胃等量蒸馏水,辛伐他汀组灌胃辛伐他汀水悬液1.7 mg/kg,当归补血汤高、中、低剂量组分别灌胃当归补血汤6 g/kg、3 g/kg、1.5 g/kg,每天1次,2周后取兔血检测VEGF和SDF-1水平,培养并鉴定兔外周血EPCs,MTT法检测增殖能力,黏附实验检测黏附能力,Transwell小室检测迁移能力,体外血管生成试剂盒检测形成小管能力。结果: 与模型组比较,辛伐他汀及当归补血汤高、中剂量组家兔血清VEGF和SDF-1水平增高(P＜0.05),且EPCs的增殖、黏附、迁移和形成小管能力均有增强(P＜0.05)。结论: 当归补血汤可能通过提高循环VEGF和SDF-1水平来促进动脉粥样硬化EPCs的活性。     

一氧化氮在VEGF介导的内皮细胞增殖与分泌效应中的作用

目的: 探讨一氧化氮在血管内皮生长因子(VEGF)介导的血管内皮细胞增殖与分泌效应中所起的作用,了解VEGF可能的作用机制。方法:将体外培养的兔主动脉内皮细胞分成对照组、VEGF处理组和VEGF+N-硝基-L-精氨酸甲酯(L-NAME)处理组,采用四氮唑盐WST-1比色法、放免法和酶联免疫双抗体夹心法分别检测吸光值及内皮素-1和Ⅷ因子辅因子水平。结果:VEGF处理组吸光值明显高于VEGF+ L-NAME处理组,且均高于对照组(P＜0.01);VEGF处理组内皮素-1和Ⅷ因子辅因子水平明显低于VEGF+L-NAME处理组,且均低于对照组(P＜0.05和P＜0.01);提示VEGF能促进内皮细胞增殖,抑制内皮细胞分泌内皮素-1和Ⅷ因子辅因子,而L-NAME能部分拮抗VEGF的上述作用。结论:一氧化氮在VEGF促进内皮细胞增殖及抑制内皮细胞分泌内皮素 -1和Ⅷ因子辅因子中起中介作用,VEGF可能部分通过一氧化氮起作用,一氧化氮是VEGF作用机制中的一个重要信号通路。