川芎嗪、葛根素、银杏黄酮及三七皂苷对鸡胚尿囊膜血管生长的影响

目的研究川芎嗪、葛根素、银杏黄酮及三七皂苷对鸡胚尿囊膜血管生长的影响。方法培养海兰种种蛋，观测局部用药对生长七日及五日鸡胚尿囊膜血管数目、血管面积的变化。结果葛根素及三七皂苷对鸡胚尿囊膜血管数目、血管面积影响不明显，川芎嗪、银杏黄酮减少鸡胚尿囊膜血管数目、血管面积，与对照组相比差异具显著性。结论川芎嗪、银杏黄酮可抑制鸡胚尿囊膜血管生长。     

黄芪、脉络宁注射液载入修饰胶原对鸡胚尿囊膜血管新生的协同促进作用

背景:课题组先期研究发现黄芪载入修饰后胶原可以促进血管新生,与生长因子载入疗效相当,运用中医理论中具有协同作用的中药合用是否能进一步提高疗效尚不清楚。目的:探查黄芪、脉络宁载入修饰后胶原促鸡胚尿囊膜血管新生及长入胶原的疗效,并证明黄芪与脉络宁是否有协同作用。方法:实验分空白组、控制组(单纯胶原)、黄芪组(黄芪注射液1mL载入胶原)、脉络宁组(脉络宁注射液1mL载入胶原)、黄芪+脉络宁组(黄芪注射液及脉络宁注射液各0.5mL载入胶原),各组植入鸡胚尿囊膜孵化7d后取出,测定鸡胚尿囊膜内微血管数、各组胶原内微血管数、血红蛋白含量、rhVEGF165阳性细胞数。结果与结论:各实验组鸡胚尿囊膜内血管呈轮辐状生长及鸡胚尿囊膜包裹标本率高于空白组与控制组,鸡胚尿囊膜内微血管数、胶原内微血管数、血红蛋白含量、rhVEGF165阳性细胞数均高于控制组,差异有显著性意义(P0.01);其中黄芪+脉络宁组又高于黄芪组与脉络宁组,差异有显著性意义(P0.05)。提示黄芪、脉络宁载入胶原后可以促进鸡胚尿囊膜内血管新生并刺激血管长入胶原内,黄芪与脉络宁合用具有协同作用,机制之一为刺激血管内皮细胞血管内皮生长因子的表达。     

数字图像分析技术在鸡胚卵黄囊膜血管形成模型中的应用

目的应用数字图像处理技术客观、准确和快速地对鸡胚卵黄囊膜(chick embryo yolk sac membrane,YSM)血管图像进行定量分析,为促血管和抗血管生成药物的评价和筛选提供有效可靠的技术支持。方法应用OPTPRO2007图像采集系统和Image-proplus6.0图像分析软件对鸡胚卵黄囊膜血管图像背景处理和血管面积及血管密度等参数进行客观、准确地测量。结果建立了有效的鸡胚卵黄囊膜数字图像血管自动分析方法和步骤并通过药物实验对鸡胚卵黄囊膜血管图像进行了参数检测和统计分析。结论应用OPTPRO2007图像采集系统和Image-proplus6.0图像处理分析软件对鸡胚卵黄囊膜图像血管进行自动分析的方法不仅操作简便而且可较准确、快速、客观地反映鸡胚血管新生情况,优于鸡胚尿囊膜血管发生模型的常规人工目测血管计数方法。     

不同鼻咽癌细胞株对鸡胚尿囊膜模型血管生成影响的比较研究

目的比较不同鼻咽癌细胞株在鸡胚尿囊膜模型上促进血管生成能力的差异。方法选用6日龄鸡胚,开窗后分别种植5-8F、6-10B及CNE-2三种鼻咽癌细胞株,三种细胞株细胞数为2×106/鸡胚者各一组,细胞数为5×105/鸡胚者各一组,另一组为对照组（PBS）,每组10个鸡胚。孵育6日后,统计分析新生血管数及血管面积/鸡胚面积比。结果种植细胞数为5×105/鸡胚时,5-8F、6-10B细胞部分鸡胚有移植瘤形成,CNE-2细胞鸡胚均未见成瘤。种植细胞数为2×106/鸡胚时,三种鼻咽癌细胞株均可100%成瘤,其新生血管数依次递减,分别为（38.7±2.50）、（33.5±4.43）、（29.7±2.71）,差异有统计学意义（P〈0.05）;血管面积/鸡胚面积比分别为（22.2±2.18）%、（18.7±2.45）%、（16.9±2.62）%,均高于对照组的（9.5±1.86）%,差异有统计学意义（P〈0.05）,且5-8F面积比高于其他两种细胞株（P〈0.05）。结论鼻咽癌5-8F、6-10B及CNE-2三种细胞株在鸡胚尿囊膜模型上血管生成能力依次减弱,实验研究可根据实际情况选用。     

加味保安方对鸡胚尿囊膜血管新生的影响

目的观察加味保安方对鸡胚尿囊膜血管(CAM)生成的影响。方法采用血清药理学方法制备含药血清。观察含药血清对培养5、6、7dCAM血管生成的影响,并与生理盐水及正常血清组对照。结果加味保安方含药血清可抑制鸡胚CAM血管生成。其中对培养5dCAM血管生成的抑制作用与生理盐水组比较差异具显著性(P<0.05);对培养6dCAM的作用,仅大剂量组与生理盐水组比较差异具显著性(P<0.05);对培养7dCAM的作用最为显著,与生理盐水组及正常血清组比较差异均具显著性(P<0.05)。结论加味保安方具有抑制鸡胚尿囊膜血管生成的作用。     

青蒿琥酯对人骨髓瘤细胞抗血管生成的作用

目的:研究青蒿琥酯对人RPMI-8226细胞诱导血管新生的抑制作用。方法:采用MTT法检测青蒿琥酯对RPMI-8226细胞增殖抑制作用;通过鸡胚绒毛尿囊膜体内血管生长实验,观察青蒿琥酯对RPMI-8226细胞诱导体内血管生成的影响;免疫蛋白印迹检测鸡胚绒毛尿囊膜内血管内皮生长因子(VEGF)和血管紧张素1(Ang-1)含量的表达变化。结果:青蒿琥酯以时间、剂量依赖方式抑制RPMI-8226细胞增殖;24h和48h后,其IC50值分别为(36.33±2.65)μmol/L和(14.31±3.28)μmol/L。在鸡胚绒毛尿囊膜体内血管生长实验中,经3、6、12μmol/L青蒿琥酯处理后,与单纯RPMI-8226细胞组比较,新生血管数目分别减少21.9%、38.2%和76.9%,差异有统计学意义;同时鸡胚绒毛尿囊膜内VEGF含量分别显著下降22.2%、34.2%和52.6%;Ang-1蛋白表达量分别显著下降15.6%、24.2%和39.6%。结论:青蒿琥酯具有抑制RPMI-8226细胞诱导血管新生的作用,其作用机制与下调RPMI-8226细胞的VEGF和Ang-1表达有关。     

青蒿琥酯对新生血管生长与成型影响的实验研究

目的通过观察新生血管生长发育与成型过程的形态学变化，研究青蒿琥酯对新生血管形成的影响。方法采用鸡胚绒毛尿囊膜模型、大鼠主动脉环无血清培养及人脐静脉内皮细胞体外培养，检测青蒿琥酯在新生血管形态发育过程中的作用。结果青蒿琥酯可影响血管内皮生长与实心条索形成，使血管生长期明显滞后，抑制血管生长。结论青蒿琥酯能通过干扰血管生长发育与成型而抑制血管生成。     

鸡胚绒毛尿囊膜模型在微血管实验研究中的应用

鸡胚的绒毛尿囊膜薄膜(CAM),作为胚胎的呼吸器官,在第4天和第5天的胚胎发展期间由被膜尿囊内脏中胚层和绒毛膜中胚层融合而成[1]。CAM是鸡胚孵化至第19天的(总期间21天)气体交换的呼吸器官,是具有丰富血管的薄膜。CAM微循环具有连续的微血管分叉状扩张[1],在生理状态下,承担适应发育中的胚胎氧需求。鸡胚CAM血管系统的发育既复杂又高度程序化。CAM血管生长于正常的鸡胚发育期间,经观察大体上经过早、中和晚三期:早期(从第5天到第7天)通过毛细血管以“出芽”方式生长;中期(从第8天到第12天)是普遍的吸收式微血管的生长,毛细血管内壁内…     

Rous肉瘤病毒引起鸡胚内脏病变的组织化学观察

一、序言 Rous和Murphy等曾将Rous肉瘤组织及上清液接种至鸡胚胚膜及胚内,引起肝,心,胃及胸腔壁长瘤及肝脏出血。以后Keogh报导Rous肉瘤活细胞于接种至鸡胚尿囊绒毛膜(CAM)后引起肝及肺产生大小瘤块;Okar-Blom及Karnofsky等分别将病毒注入胚膜均能引起胚胎肝脏出血;Duran-Reynals指出Rous病毒引起的鸡的出血病变率较成鸡为高,并认为引起出血的原因是血管内皮细胞遭受破坏和在以后出现血     

鸡胚尿囊绒毛膜法研究细胞间黏附分子-1在血管生成中的作用

目的　探讨细胞间黏附分子 1(intercellularadhesionmolecule 1,ICAM 1)在血管生成中的作用。　方法采用鸡胚尿囊绒毛膜 (chorioallantoicmembrane ,CAM)法进行在体血管生成实验。　结果　 1 10d鸡胚的尿囊绒毛膜经ICAM 1作用 3d后 ,明胶海绵周围放射状走行的微血管非常明显 ,似车辐 ,显微镜下明胶海绵内有垂直长入的微血管 ,明胶海绵周边CAM间充质内微血管数目显著多于对照组 (P <0 0 1)。 2 6d鸡胚的尿囊绒毛膜经Anti ICAM 1作用 3d后 ,明胶海绵周围放射状走行的微血管极不明显 ,显微镜下明胶海绵内几乎没有新生的微血管 ,明胶海绵周边CAM间充质内微血管数目显著少于对照组 (P <0 0 1)。　结论　结果提示 1 ICAM 1有诱导微血管生成的作用 ;2 ICAM 1参与胚胎的血管生成。     

Ultrastructural evidence for blood microvessels devoid of an endothelial cell lining in transplanted pancreatic islets.

The aim of the present study was to investigate, at the ultrastructural level, the process of revascularization of freshly isolated islets or cultured islets after transplantation under the kidney capsule of syngeneic mice. Native islets in adult pancreases from mice, pigs, and humans contained only capillaries with fenestrated endothelium. However, the endothelial cell lining was disrupted in both freshly isolated and cultured mouse islets. Shortly after transplantation (6 weeks) approximately 80% of graft microvessels contained no endothelial cell lining. Similar data on microvessel morphology were found when fetal porcine islet-like cell clusters were implanted into athymic nude mice. Re-endothelialization was a slow process, with 25% of the microvessels still lacking endothelium 6 months after transplantation of cultured mouse islets or islet-like cell cluster. However, when freshly isolated mouse islets are used only 25% of microvessels within the islet graft lacked endothelium 6 weeks after implantation. We suggest that capillaries damaged during islet isolation may provide a preformed channel, serving as a scaffold for newly formed islet graft blood vessels. The presence of non-endothelialized microvessels, with an associated lack of barrier function, might make transplanted islets more prone to thrombosis or an attack by the immune system. This provides a tentative explanation for the increased vulnerability of islet grafts when compared with whole pancreas transplants.     

Forskolin-induced differentiation of BeWo cells stimulates increased tumor growth in the chorioallantoic membrane (CAM) of the turkey (Meleagris gallopavo) egg

Invasiveness of BeWo cells has been assessed in a variety of assay systems including matrigel and mouse. At the same time BeWo cells are mostly used as model system for trophoblast fusion. Here we aimed to test the properties of BeWo cells in a combined approach. We forced BeWo cells to differentiate by culturing the cells in the presence of forskolin and then used these cells for invasion assays on the chorioallantoic membrane (CAM) of the turkey.The chorioallantoic membranes of turkey eggs were incubated with medium containing forskolin, BeWo cells cultured in medium alone, BeWo cells cultured in forskolin and washed, and BeWo cells cultured in forskolin and used directly for application. Suspensions were applied onto ten CAM per condition. For local tumor formation eggs were checked for tumor development every 24 h macroscopically for up to 12 days and immunohistochemistry for cytokeratin 18 and Ki-67 were used for further analysis.Forskolin alone did not have any deleterious effect on the CAM. When the CAM was incubated with BeWo cells cultured in medium 40% of the eggs developed a macroscopically visible tumor. BeWo cells stimulated with forskolin and washed induced tumor growth in 50% of the eggs, while forskolin stimulated BeWo cells applied directly onto the CAM induced tumor growth in 70% of the eggs. Forced differentiation of BeWo cells by forskolin may lead to syncytial fusion in a plastic culture dish. Under the conditions used here, i.e. in direct contact to a living tissue, forskolin-induced differentiation of BeWo cells leads to an increase in tumor formation in the CAM. Thus BeWo cells may use signaling pathways to decide for both differentiation pathways similar to primary trophoblast depending on the environment.     

Engineered adipose tissue supplied by functional microvessels

A volume-persistent culture of adipose tissue under in vivo conditions can be achieved only by early vascularization after cell transplantation. Cotransplantation of autologous preadipocytes with endothelial cells may enable the early formation of a capillary network. Investigations were performed in vivo in a specially adapted chorioallantoic membrane (CAM) model. Fertilized White Leghorn eggs were incubated and opened on day 3 of incubation and human dermal microvascular endothelial cell (HDMVEC) spheroids and preadipocytes were transferred in a fibrin matrix to the CAM. On day 7 after incubation the composites were explanted and immunohistologically investigated. Numerous vessels consisting of HDMVECs could be detected and the lumena of these vessels were perfused by chick erythrocytes. These results show the formation of a capillary network consisting of transplanted HDMVECs. The microcirculation of chick erythrocytes in vessels consisting of human endothelial cells proves the continuity of a newly formed capillary system to the host vessel system. The experiments demonstrate the first patent connection of tissue-engineered microvessels in adipose tissue to a host vessel system without applying exogenous angiogenic growth factors or transient transfection. The cotransplantation of endothelial cell spheroids with angiogenic mesenchymal cells may lead to the engineering of complex three-dimensional implants.     

Personalized treatment in the eradication therapy for Helicobacter pylori

Helicobacter pylori (HP) is known to be a causative bacterium of gastritis and peptic ulcers. The combination treatment consisting of a proton pump inhibitor (PPI), amoxicillin and clarithromycin (CAM) is widely used in eradication therapy, but the eradication fails in some patients. The main causes are CAM resistance of HP and individual variability in PPI metabolism related to the activity of the cytochrome P450 2C19 (CYP2C19) enzyme. In this study, we examined the usefulness of the prediction of the pharmacotherapeutic efficacy using a newly developed analysis system for HP CAM resistance and CYP2C19 genotypes. After obtaining the informed consent from 45 subjects with HP-positive peptic ulcers, biopsy specimens of the gastric mucosa were obtained by endoscopy. HP DNA extracted from the gastric mucosa was examined by the SELMAP-PCR method, the direct sequencing method or the single-nucleotide primer extension (SNuPE) method. HP detection rates by culture and the SELMAP-PCR method were 71% and 100%, respectively. Among 32 cultured HP, CAM resistance was confirmed in 6 samples by the in vitro drug susceptibility test. CAM-resistant gene mutations were also examined by the SELMAP-PCR method using 32 DNAs from cultured HP and the results were consistent with the drug susceptibility test. Among 22 patients, the eradication rate was 77%. Among 4 patients with CAM resistance determined by both the in vitro drug susceptibility test and the SNuPE method, eradication was successful in one intermediate metabolizer (IM), but not in three extensive metabolizers (EMs). Patients were divided into three groups according to their CYP2C19 phenotype: EMs, IMs and poor metabolizers (PMs). The eradication rates for 6 EMs, 12 IMs and 4 PMs were 33.3%, 91.7% and 100%, respectively. Based on these results, the information on CAM resistance in HP and CYP2C19 phenotypes in carriers could predict the pharmacotherapeutic efficacy and probability of eradication. It can then be possible to vary the dosing or to select another drug by the prediction of the pharmacotherapeutic efficacy.     

Rho and rho kinase modulation of barrier properties: cultured endothelial cells and intact microvessels of rats and mice

Previous experiments using cultured endothelial monolayers indicate that Rho-family small GTPases are involved in modulation of endothelial monolayer permeability by regulating assembly of the cellular actin filament scaffold, activity of myosin-based contractility and junctional distribution of the Ca²⁺-dependent endothelial cell adhesion molecule, VE-cadherin. We investigated these mechanisms using both cultured endothelial cells (from porcine pulmonary artery and mouse heart) and vascular endothelium in situ (mouse aorta, and individually perfused venular microvessels of mouse and rat mesentery). Exposure to Clostridium difficile toxin B (100 ng ml⁻¹) inactivated 50–90 % of all endothelial Rho proteins within 60–90 min. This was accompanied by considerable reduction of actin filament stress fibres and junctional F-actin in cultured endothelial monolayers and in mouse aortic endothelium in situ . Also, VE-cadherin became discontinuous along endothelial junctions. Inhibition of Rho kinase with Y-27632 (30 μ m ) for 90–120 min induced F-actin reduction both in vitro and in situ but did not cause redistribution or reduction of VE-cadherin staining. Perfusion of microvessels with toxin B increased basal hydraulic permeability ( L _p) but did not attenuate the transient increase in L _p of microvessels exposed to bradykinin. Perfusion of microvessels with Y-27632 (30 μ m ) for up to 100 min reduced basal L _p but did not attenuate the permeability increase induced by platelet activating factor (PAF) or bradykinin. These results show that toxin B-mediated reduction of endothelial barrier properties is due to inactivation of small GTPases other than RhoA. Rho proteins as well as RhoA-mediated contractile mechanisms are not involved in bradykinin- or PAF-induced hyperpermeability of intact microvessels.     

大鼠急性癫痫发作后齿状回分子层微血管构筑的改变

目的观测海人酸(KA)癫痫模型大鼠海马齿状回分子层微血管构筑的改变。方法采用KA癫痫模型(颈部皮下注射KA,10mg/kg),在造模后7天应用碱性磷酸酶法显示海马脑片片厚(90μm)的微血管,光镜观察,使用NIS-Element BR软件定量分析。结果海马内的微血管成层分布,构筑模式与神经元的构筑模式相一致;KA组的微血管数目明显多于对照组(P=0.003),血管平均长度明显高于对照组(P=0.000),血管平均直径无明显变化(P=0.121)。结论海马齿状回分子层微血管构筑在癫痫发病早期发生改变。     

Chick chorioallantoic membrane as an in vivo model to study vasoreactivity: characterization of development-dependent hyperemia induced by epoxyeicosatrienoic acids (EETs)

Shell-less culture of chick chorioallantoic membrane (CAM) of developing chicken embryos is a useful model to evaluate the effects of vascular agents. We assessed the response of CAM vessels to epoxyeicosatrienoic acids (EETs), derivatives of the essential fatty acid arachidonic acid, that have a number of important biological functions, including dilation of microvessels in the coronary, cerebral, renal, and mesenteric circulations. Three of four regioisomers of EETs, 14,15-, 11,12-, and 8,9-EET, induced a characteristic dose-dependent acute hyperemia within 4 min after application on 10-day-old CAMs. This response was marked in early stages of development (between days 8 and 10), but the frequency and intensity of the response were reduced after 11 days of development. Histological examination demonstrated that the hyperemia was not due to extravasation of erythrocytes. However, many capillaries were distended and contained densely packed erythrocytes as compared to uniformly arranged vessels and erythrocytes in untreated CAMs. Transmission electron microscopy showed the basal laminae surrounding capillaries remained intact, similar to those in vehicle-treated or untreated CAM tissue. The hyperemia was specific to EETs since we did not observe it to be induced by other vasodilators such as nitric oxide or prostacyclin. In conclusion, we report a novel vascular response to EETs using the CAM as an in vivo model. These lipids specifically distend a subset of capillaries in a dose- and development-dependent manner.     

In ovo assay for Marek''s disease virus and turkey herpesvirus.

Marek's disease virus (MDV) and the turkey herpesvirus (HVT) may be assayed on the chorioallantoic membrane (CAM) of the chicken embryo after intravenous inoculation of chicken embryo fibroblasts (CEF) or chicken blood leukocytes infected with these viruses. Free HVT, MDV associated with Marek's tumor cells, and lymphoblastoid cell lines derived from Marek's tumors, may be assayed in the same way. The intravenous assay is quicker than the yolk sac assay and somewhat more sensitive than in vitro or conventional CAM assay after direct inoculation of the CAM. The optimal time for inoculation was day 10 of embryo incubation; therafter the log-10 CAM lesions decreased as a negative linear function of embryo age at the time of inoculation. The log-10 CAM lesions increased as a positive linear function of the time since inoculation. The optimal time for counts was day 5 after inoculation. The log-10 CAM lesions was a linear function of the log-10 cells in the inoculum; the slope was 1.0. Venous in ovo inoculation caused as increase in the weight of the spleen proportional to the number of CAM lesions. Repression of the splenomegaly, by prior X irradiation of the embryo, did not reduce the number of CAM lesions. Embryols from lines inbred for susceptibility to Marek's disease produced more CAM lesions than embryos from resistant lines. This difference did not depend on prior exposure of the mothers to MDV or HVT.     

A modified chorioallantoic membrane (CAM) assay for qualitative and quantitative study of growth factors

Summary The application of Thermanox tissue culture coverslips to the day 9 CAM of the chick causes constant effects beneath the carrier after 3 days, and these are associated with a change in the blood vessel pattern. Histological sections show enormous thickening of the CAM in the reactive areas. The stroma of the CAM shows fibrocyte proliferation, leucocyte infiltration, and clusters of dispersed ectodermal epithelial cells exhibiting signs of necrosis. The latter obviously cause a strong vascular response. The same effects are seen when the Thermanox discs are applied at day 11. Following application on day 12 a positive or negative response to the carrier is observed, whereas on day 13 no such carrier effects are seen. The only remaining effect is compression of the intra-ectodermal capillary plexus of the CAM. This can macroscopically be seen after peroxidase staining of the blood vessels. The effect of 5 l PBS dried on the Thermanox disc and applied to the day 13 CAM is to cause, after 3 days, hyperosmotic damage to the ectodermal epithelium, which becomes overgrown by fibrocytes. We found dose-dependent effects of salt-free human bFGF applied to the day 13 CAM. The first and main effect is fibrocyte proliferation (0.5 g). New capillaries appear with higher doses, but are not as frequent as would be expected for an angiogenic substance (1.25–2.5 g). Also with higher doses additional hyperplasia of the endodermal (3.75 g) and ectodermal (5 g) epithelium can be seen. The latter might be a non-specific hyperosmotic effect. Leucocytes are regularly present within the reactive areas. When salt-free angiogenin is applied to the day 13 CAM, some effects appear with doses of 4.6 g and more. The ectodermal epithelium of the reactive areas is discontinuous, exhibiting signs of necrosis. It is overgrown by parallel fibrocytes. Whether this is a non-specific hyperosmotic effect, or indicates enhancement of invasive growth, calls for further investigation.