在鸡胚绒毛尿囊膜上评价血管生成技术的应用与优化

目的：用鸡胚绒毛尿囊膜（CAM）评价血管生成并进行优化,为抗血管生成药物筛选提供良好的体内实验模型。方法：对CAM技术的各个环节,包括合适日龄胚蛋选择、蛋壳开窗及暴露CAM的方法、载体选择、结果分析等进行了实验对比。结果：采取气室开窗,以甲基纤维素为药物载体,促进血管生成的药物在孵化11d以后、抑制血管生成的药物在孵化7～9d给药,鸡胚存活率为85.8%,生长状况良好,被检测药物有明显的抑制或促进血管生成的效应。结论：通过优化建立了操作简便、鸡胚存活率高的在CAM上评价血管生成的技术;应用CAM技术评价了若干影响血管生成的因子。     

鸡胚模型在生物研究中的应用进展

实验动物模型在预防、诊断、治疗疾病和探讨疾病的发生机制等方面起到了至关重要的作用。鸡胚发育过程清楚,利用鸡胚本身的结构特点,可作为研究与胚胎发育相关的生物学实验模型。另外,鸡胚绒毛尿囊膜（CAM）血管丰富,是天然免疫缺陷宿主,可作为血管药理学、肿瘤学等方面研究的一个较为理想的实验模型。本文综述了鸡胚模型在生物实验研究中的应用进展。     

Canstatin induces apoptosis in gastric cancer xenograft growth in mice through the mitochondrial apoptotic pathway

Canstatin, the non-collagenous domain of collagen type IV α-chains, belongs to a series of collagen-derived angiogenic inhibitors. In this study, the inhibitory effect of recombinant canstatin on tumour growth was investigated using a gastric cancer xenograft model. The volume and weight of tumours in mice treated with canstatin were lower than that in mice treated with PBS. Accordingly, the survival rate of these mice was significantly higher than that of mice bearing tumours treated with PBS. Moreover, valuable insight into the mechanisms mediated by canstatin was obtained.     

Alg-g-PEI/pVEGF复合物体内促血管生成作用

研究氧化海藻酸——低分子量聚乙烯亚胺两性刷型共聚物 (Algin-a-te-graft-PEI, Alg-g-PEI) 与血管内皮生长因子质粒 (pVEGF) 复合后对体内血管生成的影响。采用细胞和斑马鱼实验检测了Alg-g-PEI/pVEGF复合物的毒性;通过凝胶电泳实验评价了Alg-g-PEI对DNA的保护作用;利用鸡胚绒毛尿囊膜 (CAM) 和斑马鱼模型,选用PEI 25K/pVEGF作为阳性对照、生理盐水为空白对照,观察复合物对血管新生的促进作用。结果发现：Alg-g-PEI对质粒有较好的保护作用;Alg-g-PEI/pVEGF复合物具有较低的细胞、斑马鱼毒性,能显著促进CAM和斑马鱼的血管生成,且具有剂量依赖性,当pVEGF用量等于2.4 μg/CAM时,复合物对CAM血管生成的促进作用最明显,血管面积和CAM面积比 (VA/CAM) 为44.04％,高于阳性对照组 (35.90％) 和空白对照组 (24.03％) (**P＜0.01)。复合物对斑马鱼血管新生的促进作用随氮磷比 (N/P) 的增加而增强,其中N/P=110时,血管新生最明显：肠下血管总长度和面积分别为1.11 mm和1.70×103像素,高于空白对照组 (0.69 mm和0.95×103像素) (**P＜0.01) 和阳性对照组 (0.82 mm和1.11×103像素) (**P＜0.01) 。综上所述,Alg-g-PEI/pVEGF能于体内促进血管生成,该载体有望用于临床缺血性疾病的治疗。     

The effect of early prenatal hypercapnia on the vascular network in the chorioallantoic membrane of the chicken embryo

Over the last decade, the poultry sector has sought to develop novel ways to monitor chicken embryonic growth, health, and quality to control and optimize egg incubation conditions, particularly the concentration of dissolved gases (O(2), CO(2)). One of the parameters, which may change under different gas concentrations, is the angiogenesis in the chorioallantoic membrane (CAM), the organ for gas exchange of the chicken embryo. In this study, a newly developed methodology was used to quantify the angiogenesis in the CAM under normal and early hypercapnic conditions (i.e., increased CO(2) concentrations). Two experiments were conducted in which the same CO(2) profile was applied. The development of the vascular system was monitored from embryonic day (ED) 10 until ED 14 in Experiment 1, and until ED 16 in Experiment 2. This development was characterized by two different parameters-the vascular fraction (VF) as a measure for the density of the vascular network and the fractal dimension (FD) as a measure for the degree of branching of the vascular network. Moreover, in Experiment 2, embryo weights were compared between both groups. The proposed methodology showed that differences in the development of the vascular system could be observed across groups but also as function of the ED. Both VF and FD and the embryo weights were shown to be higher in the hypercapnia group compared to the control group.     

Proper autophagy is indispensable for angiogenesis during chick embryo development

People have known that autophagy plays a very important role in many physiological and pathological events. But the role of autophagy on embryonic angiogenesis still remains obscure. In this study, we demonstrated that Atg7, Atg8 and Beclin1 were expressed in the plexus vessels of angiogenesis at chick yolk sac membrane and chorioallantoic membrane. Interfering in autophagy with autophagy inducer or inhibitor could restrict the angiogenesis in vivo, which might be driven by the disorder of angiogenesis-related gene expressions, and also lead to embryonic hemorrhage, which was due to imperfection cell junctions in endothelial cells including abnormal expressions of tight junction, adheren junction and desmosome genes. Using HUVECs, we revealed that cell viability and migration ability changed with the alteration of cell autophagy exposed to RAPA or 3-MA. Interestingly, tube formation assay showed that HUVECs ability of tube formation altered with the change of Atg5, Atg7 and Atg8 manipulated by the transfection of their corresponding siRNA or plasmids. Moreover, the lost cell polarity labeled by F-actin and the absenced β-catenin in RAPA-treated and 3-MA-treated cell membrane implied intracellular cytoskeleton alteration was induced by the activation and depression of autophagy. Taken together, our current experimental data reveal that autophagy is really involved in regulating angiogenesis during embryo development.     

Increased expression of VE-cadherin correlates temporally with differentiation of a restrictive endothelial barrier during normal angiogenesis in vivo

The purpose of this study was to evaluate temporal expression of VE- and N-cadherins within the angiogenic chick chorioallantoic membrane (CAM). Whether their relative patterns of expression changed in conjunction with abrupt differentiation of the restrictive CAM endothelial barrier between days 4.5 and 5.0 of the 21 days gestation was evaluated. Immunoblots against VE-cadherin depicted an increase of VE-cadherin expression between days 4.5 and 5.0, but no change in expression was detected between days 5.0 and 6.0. N-cadherin expression, on the other hand, remained uniform from day 4.5 to day 6.0. Immunogold-labeled anti-VE-cadherin was found exclusively on the CAM endothelium, and principally along the lateral inter-endothelial junctions. Hence, VE-cadherin expression by the angiogenic endothelium was similar to that of adult endothelium. That VE-cadherin expression by the CAM endothelium was increased between days 4.5 and 5.0 serves to suggest a temporal correlation with the ontogeny of restrictive barrier function in angiogenic endothelium in vivo.     

蝎毒多肽提取物的抗血管生成作用

用不同浓度的东亚钳蝎素的多肽提取物PESV(4～20μg／ml)作用于人脐静脉内皮细胞(HUVEC),观察HUVEC增殖活性和凋亡变化,增殖活性检测采用BrdU掺入的ELISA法,凋亡水平和凋亡相关基因Bcl-2和Bax表达的检测采用流式细胞术检测;用鸡胚尿囊膜(CAM)显示PESV对血管生成的抑制作用。结果显示,PESV抑制HUVEC的增殖,而对乳腺癌细胞MDA-MB-231的增殖无明显影响;PESV作用72h后,HUVEC凋亡相关基因Bcl-2表达降低,Bax表达增加,凋亡细胞比例增至10．5％,明显高于对照组;0．5mgPESV能明显抑制CAM新生血管的形成。因此,PESV具有良好的体外抗肿瘤血管生成活性,PESV作为一种肿瘤血管抑制剂的天然药物来源,其有效成分和药理作用有待进一步研究。     

The potential of spatially resolved spectroscopy for monitoring angiogenesis in the chorioallantoic membrane

Over the last decade, the poultry sector has sought to develop ways to monitor chicken embryonic development as to optimize the incubation conditions. One of the parameters of development which may change under different incubation conditions is the angiogenesis in the chorioallantoic membrane (CAM). To be able to quantify these changes in the angiogenesis and detect long-term effects on health, a non-destructive technique is necessary. In this article, the first steps toward such a non-destructive technique are successfully taken. A spatially resolved spectroscopy set-up is built and tested for its potential to measure changes in angiogenesis with incubation time, and differences between a normal and hypercapnic incubation. In this first study, reflectance measurements are performed directly on the CAM as the eggshell considerably complicates the analysis. This issue should be addressed in future research to come to a really non-destructive technique. An experiment was conducted in which one group was incubated under normal conditions, and another under early prenatal hypercapnic conditions (i.e., increased CO(2) concentrations). The angiogenesis in the CAM was measured at embryonic day (ED) 10, 13, and 16. The measurements showed a clear blood spectrum with an increasing amount of blood in time, and significant differences in the reflectance as function of the source-detector distances. However, no significant differences between the hypercapnia and the control group could be detected.     

Antiangiogenic effect of sulphated and nonsulphated glycosaminoglycans and polysaccharides in the chick embryo chorioallantoic membrane

The inhibiting effect of sulphated and nonsulphated glycosaminoglycans and polysaccharides on the normal outgrowth of capillaries was tested in the chick embryo chorioallantoic membrane (CAM) with and without the presence of hydrocortisone. An antiangiogenic response to 50 µg of heparin and heparan sulphate (without hydrocortisone present) was observed in 38.8% and 23.1% of the CAMS, respectively, while the antiangiogenic response rate for dermatan sulphate, chondroitin sulphate A or C, hyaluronic acid and keratan sulphate was 15.9–0%. All sulphated homopolysaccharides tested were more effective than the naturally occurring glycosaminoglycans. Nonsulphated dextran and (methyl) cellulose had no antiangiogenic effect, while largely desulphated heparin retained such an effect. Hydrocortisone generally improved the antiangiogenic effect, a 100% response was obtained when it was combined with cellulose sulphate or fucoidan (polyfucose sulphate derived from marine algae), but the antiangiogenic effect of the largely desulphated heparin was unaffected by the presence of hydrocortisone. The results show that different polysulphated polysaccharides also have an antiangiogenic effect, without the addition of corticosteroids. The effect was apparently independent of their degree of sulphation, but the glycosidic structure may be of critical importance.     

肿瘤干细胞与表观遗传学调控

关于恶性肿瘤发生、复发与转移机制的研究由来已久,但目前的临床治疗方法依然不能克服肿瘤复发与转移的难题,肿瘤患者的生存率并未得到显著改善。近年来的研究提示肿瘤的起源、复发与转移的真正原因可能是存在于肿瘤内的极少数具有干细胞特性的细胞,即肿瘤干细胞（cancer stem cells,CSC）。与此同时,越来越多的研究表明,对于肿瘤干细胞的发生与功能维持,表观遗传学的调控机制可能发挥着极其重要的作用。该文简要综述目前肿瘤干细胞和表观遗传学相关领域的研究进展,并对肿瘤干细胞形成及发展过程中表观遗传学的调控作用及机制进行重点介绍。