Introduction of DT40 cells into chick embryos

Aim: To examine the transfection of exogenous genes into chick embryos, applying the characteristics of avianleukosis vires (ALV)-induced chicken B cell line DT40 to the production of chimeric birds. Methods: The DT40cells incorporated with exogenous gene (lacZ constructs encoding Escherichia coli β-galactosidase: β-gal) were intro-duced into chick embryos by the injection of cells into stage X blastoderm. Manipulated eggs were incubated for 3 (trial1) or 6 (trial 2) days, and the expression of lacZ DNA was detected by a histochemical staining method of β-galactosi-dase and polymerase chain reaction (PCR) analysis. Results: The survival rates of the manipulated embryos incu-bated for 3 days (stage 18-20: trial 1) and 6 days (stage 28, 30: trial 2) were about 42% and 38%, respectively.The expression rates of the lacZ gene in the embryos in the trials 1 and 2 were about 60% and 23%, respectively, forthe survived embryos. Conclusion: The rate of embryonic viability and expression rate of introduced genes were notso high, but it suggested the possibility of utilizing the DT40 cells as a vector for carrying exogenous genes into chickembryos. (Asian J Androl 2001; 3: 49-53)

Manganese enhances the expression of the manganese superoxide dismutase in cultured primary chick embryonic myocardial cells

In the present study, the effect of manganese（Mn） on antioxidant status and the expression of the manganese superoxide dismutase（MnSOD） gene in cultured primary myocardial cells collected from the chick embryos was investigated. The hypothesis that Mn supplementation would enhance the expression of MnSOD in cultured primary myocardial cells of chick embryos was tested. Eggs collected from Mn-depleted Arbor Acres laying breeder hens were incubated for 10 days and then myocardial cells were isolated and cultivated for 8 days. The embryonic myocardial cells on day 6 were treated with Mn in the cell culture medium at different time points when the proportion of cells showing spontaneous contraction was over 95% after the 3-day primary culture. A completely randomized design involving a 3 Mn levels（0, 0.5 and 1.0 mmol L^（-1））×3 incubation time points（12, 24 and 48 h） factorial arrangement of treatments（n=6） was used in the current experiment. The results showed that MnSOD activity and m RNA expression level were induced by Mn and increased with incubation time, which supported the hypothesis that Mn would enhance the expression of the MnSOD gene, and thus might protect myocardial cells from oxidative stress during the chick embryonic development.

Ameliorating Effects of Thyroxine and Atropine in Phosphamidon Intoxicated Chick Embryos

The effects of thyroxine and atropine in ameliorating phosphamidon intoxication in chick embryos was studied. Treatment of phosphamidon significantly enhanced the moriality and abnormalityrates, decreased the average body weights, and cholinesterase activity in chick embryos. When thyroxine was administered to the phosphamidon intoxicated embryos, the above parameters changedsignificantly, indicating an ameliorating effect of thyroxine against phosphamidon intoxication in chick embryos. The combined thyroxine and atropine therapy did not further improve the ameliorating effect. Since in many respects chick embryo development parallels that of mammalian embryos,a short-term use of thyroxine as a protective agent against organophosphate toxicity might be useful

Lens Capsule HSPG-Perlecan Regulates Lens Fibre Differentiation during Chick Embryo Development

Lens fibre differentiation is a life-long process related with lens transparency, and is particularly intense during development, being related with an FGF-2 antero-posterior gradient at the equator level as the main growth factor involved which has been related with the basal membrane of the lens anlagen known as “Lens capsule”. However the lens fibre differentiation induced by FGF2 depends, as in other biological systems, on the local bioavailability of FGF-2 regulated by their relationship with extracellular matrix molecules as Heparan Sulphate Proteoglycans. Here, we try to clarify how Perlecan (a heparan sulphate proteoglycan specific from basement membranes) is involved in lens fibre differentiation at earliest stages of eye development. Our results show that Perlecan, is a major component in the lens capsule during the earliest stages of lens development in chick embryos being present during lens plate induction, lens vesicle stage and the onset of lens fibre differentiation. In order to demonstrate a direct involvement of HSPG-Perlecan in lens fibre differentiation, we generate depleted lenses by HSPG-Perlecan synthesis disruption and specific enzymatic digestion. The HSPG-Perlecan depleted lens show a significant delay or abolition in the lens fibre differentiation which remains in an immature cells displaying DNA synthesis in the posterior epithelium and a decrease in FGF2 lens expression. These data support the hypothesis that lens capsule HSPG-Perlecan is a key molecule involved in lens fibre differentiation during development, probably by involvement in FGF-2 biodisponibility.

Antioxidant systems in chick embryo development. Part 1.Vitamin E,carotenoids and selenium

Chick viability is known to be an important factor determining profitability of the poultry industry. Chick embryo tissues contain a high proportion of highly polyunsaturated fatty acids in the lipid fraction and therefore need antioxidant defence. The antioxidant system of the developing embryo and newly hatched chick includes the antioxidant enzymes(superoxide dismutase, glutathione peroxidase, catalase), water-soluble antioxidants(ascorbic acid, taurine, carnitine, glutathione, etc.), fat-soluble antioxidants(vitamin E, carotenoids, coenzyme Q) as well as selenium(Se). In fact, the high levels of endogenous antioxidants within the egg and embryonic tissues can clearly serve as a major adaptive mechanism for the protection of the tissue during the oxidative stress experienced at hatching. It has been shown that among different nutrients in the maternal diet which could significantly affect chick embryo development and their viability in the early posthatch life, natural antioxidants have been suggested to play a central role. Our data indicate that increased supplementation of the maternal diet can substantially increase concentrations of vitamin E, carotenoids(especially canthaxanthin) and Se in developing chick tissues and significantly decrease susceptibility to lipid peroxidation being effective nutritional tools to deal with various commercial stresses in poultry production.

Proliferation of exogenously injected primordial germ cells (PGCs) into busulfan-treated chicken embryos

Aim: This study was designed to investigate the effect of busulfan treatment on the proliferation of chicken primordialgerm cells (PGCs) in vivo, focusing on the preferential settlement of PGCs onto the germinal ridges of chicken em-bryos. Methods: Busulfan (250 ng/egg) was injected into the egg white of freshly oviposited fertilized eggs, whichwere then incubated. Embryonic development and viability were examined, and exogenous PGCs collected from embry-onic blood vessels were injected into the germinal crescent region of recipient embryos. The number of PGCs residedonto germinal ridges of the right and left sides were compared. Results: Busulfan had a slight harmful effect on theembryo viability and the PGCs proliferation. The number of PGCs resided onto the left side of germinal ridges wasslightly higher as compared with the right side. Conclusion: Busulfan suppressed the viability of embryos and the pro-liferation of endogenous PGCs in the recipient embryos. However, the number of exogenous PGCs proliferated washigher in embryos treated with busulfan than those without busulfan. Data also suggest the possibility of a preferentialresidence of PGCs toward the left side of the germinal crescent region as compared with the fight, which may be due toa more advanced functional development of the left gonad than the right. (Asian J Androl 1999 Dec; 1: 187-190)

Research on the appropriate way to transfer exogenous substances into chicken embryos

In biological research, chicken embryos are a classic experimental model for the exploration of the embryonic development and cell differentiation. Transferring exogenous substances into chicken embryos for producing medical antibodies has been widely used in the production practice. However, there are few studies about the effect of the different injection site and dosage on chicken embryos. The aim of this study was to explore the effects of different injection sites and dosages on chicken embryo hatching rate and development, so as to provide a basis for further studies using the chicken embryo model. Freshly laid eggs （Rugao yellow chicken） were injected with different doses of saline at the tip, equatorial plane and the blunt end of the egg shell, respectively. Egg hatching rate was recorded and compared among injection sites and different doses. A trypan blue stain was also injected at the aforementioned sites and the growth of chicken embryos was observed. The SPSS （statistical package for the social science） software was used to analyze the relationship between the chicken eggs hatching rate and the different injection sites or the different dosages. The experimental results showed that there were significant differences on egg hatching rates among the different injection sites and doses （P〈0.05）. The hatchability of the blunt end injection group was significantly higher than that of the other two sites. The egg hatching rate decreased with increased saline doses. The egg hatching rate of the 100 pL saline injection group was higher than the 200 and 300 μL dosage groups. Ultimately, we suggest that the optimal chicken embryo injection process is during early development, at the blunt end site with a dose less than 100 μL to minimize damage to the egg.

Spontaneous and light-induced photon emission from intact brains of chick embryos

Photon emission (PE) and light-induced photon emission(LPE) of intact brains isolated from chick embryos have been measured by using the single photon counting device. Experimental results showed that the intensi-ty level of photon emission was detected to be higher from intact brain than from the medium in which the brain was immerged during measuring, and the emission intensity was related to the developmental stages, the healthy situation of the measured embryos, and the freshness of isolated brains as well. After white light illumination, a short-life de-layed emission from intact brains was observed, and its relaxation behavior followed a hyperbolic rather than an expo-nential law. According to the hypothesis of biophoton emission originating from a delocalized coherent electromagnetic field and Frohlich’s idea of coherent long-range interactions in biological systems, discussions were made on the signifi-cance of photon emission in studying cell communication, biological regulation, living system’

ULTRASTRUCTURAL MORPHOLOGY OF THE E_tRFC IN LYMPHOID CELLS DURING YOLKSAC HEMOPOIESIS IN CHICK EMBRYO

The previous report has shown that some lymphoid cells were found in the blood of chick embryo after incubation for 48, 58 and 68 h, were capable of forming E rosettes in conjunction with sheep red blood cell (SRBC), and showed a positive reac-

Visualization of bHLH transcription factor interactions in living mammalian cell nuclei and developing chicken neural tube by FRET

基本 helix-loop-helix (bHLH ) 的成员基因家庭在脊椎动物神经发生起重要作用。在这研究，共焦的基于显微镜学的荧光回声精力转移(烦恼) 被用来在各种各样的生理的条件下面监视 bHLH 蛋白质蛋白质相互作用。织物特定的 bHLH 使活跃之物， NeuroD1， Mash1， Neurogenin1 (Ngn1 ) ， Neurogenin2 (Ngn2 ) ，和无所不在的表示 E47 蛋白质与提高的黄荧光蛋白质(EYFP ) 被标注并且分别地提高了青色荧光蛋白质(ECFP ) 。潜水艇 bHLH 熔化蛋白质的细胞的本地化和活动性在 HEK293 房间被检验。由短暂 transfection 并且在 ovo electroporation，四织物特定的 bHLH 使活跃之物和 E47 蛋白质在 HEK293 房间和发展中的小鸡胚胎是过去表示的神经试管。与漂白的领受人相片方法，烦恼能在 transfected 的原子核在这些 bHLH 蛋白质对之间被检测房间和发展中的神经试管。Mash1/E47 和 Ngn2/E47 烦恼对更高出现烦恼效率在中间并且小鸡胚胎的侧面的一半神经试管分别地。它建议这些 bHLH 蛋白质对在特定的区域与他们的下游的目标基因的规章的元素形成了功能的 DNA 蛋白质建筑群。这个工作将帮助一个在 vivo 理解 bHLH 因素的行为。

Effect of Thyroid Hormone Deficiency on Tubulin Synthesis in Developing Chick Brain

<正> Thyroid hormone deficiency was created artificially in chick embryos by injecting propyl-thiouracil(PTU).The tubulin was quantified by ~3H-colchicine assay(expressed as colchicine bindingactivity).Thyroid hormone deficiency resulted in 16％ to 28％ decline of the tubulin level in thedeveloping chick brain from 15 to 19 days of embryonic age as compared with the control,whereasthe total protein amount was not significantly affected.It suggests that thyroid hormone specificallyaffected the synthesis of brain tubulin.

神经母细胞瘤动物模型研究进展与应用

神经母细胞瘤(neuroblastoma,NB)是儿童最常见的实体恶性肿瘤,居我国儿童肿瘤发病率第四位,占儿童肿瘤死亡人数的15%,高危患者存活率低。目前对于NB的发病及药物治疗机制知之甚少。NB动物模型可以表征NB发展特征,是研究预防和治疗NB的重要工具,然而尚未有一种动物模型可以模拟人类NB的所有特征。本文介绍了当前研究较多的几种NB动物模型(小鼠模型、鸡胚绒毛尿囊膜模型和斑马鱼模型),并对每种NB动物模型的种类、构建方法、特征、优缺点及研究进展做了详细阐述,同时对NB的应用方向及前景进行概括,以期为NB动物模型构建和NB治疗等提供理论依据。

人参皂甙Rg3抑制肿瘤新生血管形成的研究

目的 :探讨人参皂甙 Rg3抗肿瘤生长的作用机制。方法 :利用鸡胚绒毛尿囊膜 (CAM)观察 Rg3作用后肿瘤新生血管的生长情况 ,并采用 L ewis肺癌模型进一步探讨 Rg3在体内对肿瘤生长及肿瘤新生血管形成的影响。结果 :Rg3浓度为 0 .1和 0 .5 mm ol/L时 ,CAM血管生长抑制指数分别达 (6 2 .4± 9.3) %和 (4 5 .6± 5 .7) % ,与对照组相比差异非常显著 (P<0 .0 1)。对 L ewis肺癌荷瘤小鼠分别以 5、10、2 0 mg/kg的剂量隔日灌胃一次给予 Rg3,连续 10次 ,2 0 d后观察到 3组的肿瘤瘤质量分别为 (1.77± 0 .2 1)、(1.5 8± 0 .17)及 (1.2 2± 0 .2 9) g,与对照组比较有显著性差异 (P <0 .0 1) ,生长抑制率分别为 2 3.0 4%、31.30 %和 47.10 % ,同时观察到 Rg3组的新生血管数也明显减少。 结论 :人参皂甙 Rg3可明显抑制 L ewis肺癌的生长 。

通心络促血管生成作用的实验研究

目的 探讨中药通心络对鸡胚绒毛尿囊膜 (CAM)模型血管生成作用的影响。方法 运用血清药理学的方法制备药物血清 ,鸡胚随机分为空白血清组、贝复济组及通心络大、小剂量组 ,检测尿囊膜的血管增生程度。结果 通心络大剂量组CAM的血管增生数明显高于空白血清组 (P <0 .0 5) ,而与贝复济组无明显差异 (P >0 .0 5) ;通心络小剂量组与空白血清组相比无明显差异 (P >0 .0 5)。结论 通心络可促进鸡胚绒毛尿囊膜的血管增生 。

黄芪多糖、淫羊藿多糖和淫羊藿总黄酮对新城疫病毒感染细胞的影响

将黄芪多糖 (APS)、淫羊藿多糖 (EPS)、淫羊藿总黄酮 (EF)分别与新城疫病毒 (NDV)Ⅰ、Ⅳ系以 3种顺序加入到培养 2 4h的鸡胚成纤维细胞 (CEF)中 ,即先加中药后接种病毒、先接种病毒后加中药、病毒和中药混合感作后同时加入。于病毒接种后 72h测定NDVⅠ系的半数组织培养感染剂量 (TCID50 )和NDVⅣ系的血凝效价 ,以评价 3种中药成分对NDV感染细胞的影响。结果表明 ,APS和EPS仅在先于病毒加入时对NDV有抑制作用 ,而EF无论以何种方式给药均呈抑制作用。提示它们有一定的抗病毒作用 。

10种中药成分对单层鸡胚成纤维细胞增殖的影响

将安全浓度范围内的黄芪多糖、当归多糖、蜂胶多糖、淫羊藿多糖、板蓝根多糖、黄芪黄酮、蜂胶黄酮、淫羊藿黄酮、黄芪皂苷和人参皂苷等 10种中药成分分别加入已培养 2 4h、刚形成单层的鸡胚成纤维细胞 (CEF)中 ,在加药后12 ,2 4 ,36 ,4 8和 6 0h用MTT法测定它们对CEF增殖的影响。结果表明 ,黄芪皂苷、黄芪多糖、板蓝根多糖和蜂胶黄酮主要表现为促进增殖 ;淫羊藿多糖主要具抑制效应 ;其他 5种中药成分在某些浓度和时间点能促进增殖 ,而在某些浓度和时间点则抑制增殖 ,并有一定的量效和时效关系。

鸡肠上皮细胞的分离及原代培养方法

取18日龄鸡胚，研究鸡肠上皮细胞（IEC）分离及原代培养的方法。结果表明：较好的分离条件是肠组织经0.1g／L中性蛋白酶和300U／mL胶原酶Ⅺ联合消化；较佳的培养条件是细胞在含2．5％～5％胎牛血清的DMEM培养基中，39℃、5％～7．5％CO2下培养，IEC可在1～2d贴壁，6～7d明显增殖，11～12d汇合成片。

黄芪多糖和淫羊藿多糖对培养细胞增殖和衰老的影响

将安全浓度范围内的黄芪多糖(astragaluspolysaccharide,APS)和淫羊藿多糖(epimediumpolysaccharide,EPS)分别加入到培养12h和24h的鸡胚成纤维细胞(CEF)中,测定加药后CEF增殖率和成层率的动态变化。结果表明,2种多糖均能促进细胞增殖和延缓细胞衰老,EPS的作用强于APS,具有一定的量效、时效关系。

几种天然药物成分在体外CEF中最大安全浓度的测定

采用组织细胞培养方法 ,测试了板蓝根多糖、当归多糖、黄芪多糖、黄芪皂甙、人参皂甙、淫羊藿多糖、蜂胶多糖、蜂胶黄酮、黄芪黄酮、淫羊藿黄酮共 1 0种天然药物成分在鸡胚成纤维细胞 ( CEF)体外培养中的最大安全浓度。结果表明 :黄芪多糖、当归多糖对鸡胚成纤维细胞毒性最低 ,最大安全浓度达 1 562 .5μg/ m L ,其后依次是板蓝根多糖、人参皂甙、黄芪黄酮、黄芪皂甙、蜂胶多糖、淫羊藿多糖、淫羊藿黄酮和蜂胶黄酮 ;蜂胶黄酮的毒性最大 ,其最大安全浓度仅 4.883 μg/ m L。

华蟾素联合三氧化二砷抑制鸡胚尿囊膜血管生成的实验研究

目的:观察和比较华蟾素注射液、三氧化二砷注射液单药以及联合用药后对鸡胚尿囊膜(CAM)血管生成的抑制作用。方法:100只七日龄鸡胚随机分为4组(每组25只):生理盐水(NS)对照组、华蟾素组、As2O3组和华蟾素联合As2O3组。CAM上植入甲基纤维素碟,加入各组药物,观察对血管生长的影响。结果:华蟾素、As2O3以及两药联合对鸡胚尿囊膜新生血管均有抑制作用,以两药联合后抑制血管的作用最强。结论:华蟾素、As2O3以及两药联合均具有抗血管生成作用,两药联用后且具有协同作用。