鸡胚成纤维细胞的制备、维持及传代

在禽类的原代细胞培养中，鸡娅成纤维细胞（chicken emhry fi—broblast，CEF）得到普遍应用。CEF的培养是在一定的条件下，在体外模拟体内生理环境，使从体内取出的成纤维组织细胞生存、生长和传代，并维持原有组织细胞的结构和功能特性。早年的组织培养工作者，如Carrel等曾用鸡胚做过大量研究工作。     

堆型艾美耳球虫早熟减毒株裂殖子的细胞培养

以收集于雏鸡十二指肠的堆型艾美耳球虫早熟减毒株裂殖子接种于鸡胚成纤维细胞、鸡胚肝细胞、鸡胚小肠上皮细胞和雏鸡肾细胞，４１℃培养１２０小时，其在鸡胚成纤维细胞和鸡胚肝细胞中不能继续发育，在鸡胚小肠上皮细胞和雏鸡肾细胞中可发育到卵囊阶段。     

不同pH值对鸡胚成纤维细胞培养的影响

利用不同的pH对鸡胚成纤维细胞进行培养,观察细胞在不同pH条件下的生长状况,研究不同pH对于细胞培养的影响。设置pH值为6.4、6.8、7.2、7.4、7.6和7.8的六个培养条件分别对鸡胚成纤维细胞进行培养,同时做重复对照,给每组中生长良好的细胞接毒,观察其接毒后在不同pH下的生长状况。通过观察可以看出细胞的耐酸性要强于耐碱性,而且鸡胚成纤维细胞在7.4～7.6的范围内生长情况最为良好和稳定。     

室温消化法制备鸡胚成纤维细胞的试验研究

鸡胚组织是组织培养最早被利用的对象。早年组织培养工作者，如Carrel等曾用鸡胚做过大量研究工作。目前，鸡胚成纤维细胞已被广泛应用于细胞和分子生物学研究的许多方面，这主要是由于成纤维细胞是最易培养的细胞，其良好的耐受性使之易于进行从基因转染到微注射等较多领域的研究。本试验经长期摸索、比较、找到一种制备鸡胚成纤维细胞的优化方法——室温消化法。     

小鼠胚胎干细胞裂解液诱导鸡胚成纤维细胞发生重编程的研究

本研究采用小鼠胚胎干细胞(ESCs)裂解液与鸡胚成纤维细胞共孵育,通过形态学观察和多能性检测,旨在探讨小鼠ESCs裂解液逆转化鸡胚成纤维细胞为多能干细胞的可行性。结果表明:与小鼠ESCs裂解液共孵育的鸡胚成纤维细胞均变为圆形细胞,形成了42.33个细胞集落;经AKP染色以及Oct-4和SSEA-1免疫细胞化学染色,均呈阳性反应,表现出一定的多能干细胞特性;经核型分析表明这些细胞来源于鸡胚成纤维细胞。单独的重编程操作过程和与鸡胚成纤维细胞裂解液共孵育均无法将鸡胚成纤维细胞重编程为具有ESCs特征的细胞,说明参与重编程鸡胚成纤维细胞的物质来源于小鼠ESCs裂解液。因此,ESCs裂解液诱导体细胞重编程逆转化为多能干细胞的作用能够在小鼠和鸡之间发生。     

采用继代鸡胚成纤维细胞生产鸡传染性法氏囊细胞苗的试验

采用原代鸡胚成纤维细胞和继代鸡胚成纤维细胞,生产了鸡传染性法氏囊细胞苗,效价(TCID50/mL)无明显差异.采用继代鸡胚成纤维细胞生产鸡传染性法氏囊细胞苗,减少种蛋消耗,降低劳动强度,增加产量,降低生产成本.     

一种优化鸡胚成纤维细胞制备方法及生长曲线测定

鸡胚成纤维细胞（CEF）的来源方便,制备简单,耐受性好,且适合于许多病毒的生长繁殖,因此被广泛用于疫苗的生产、病毒的培养以及一些细胞和分子生物学的研究。然而传统的鸡胚成纤维细胞制备方法存在制备时间长、死细胞较多和细胞贴壁慢的问题。笔者根据经验摸索出一种实验室快速制备CEF的简便方法,并通过台盼蓝染色用细胞计数法绘制了原代CEF及次代CEF的生长曲线。该简便方法的建立为实验室快速制备高质量高纯度的鸡胚成纤维细胞提供了方便,同时对其生长曲线的测定也为进行基因转染方面的研究积累了资料。     

利用不同方法培养鸡胚成纤维细胞的研究

采用胰酶消化法和组织块培养法培养了大量优质的鸡胚成纤维细胞,并对培养的细胞进行了冻存和复苏,建立了一套较完备、快速的鸡胚胎成纤维细胞的培养模式。并探讨这两种培养方法的优缺点。     

鸡新城疫病毒致鸡胚成纤维细胞病变特性研究

目的:研究鸡新城疫病毒致鸡胚成纤维细胞病变特性.方法:在成功制备鸡胚成纤维细胞单层的基础上,接种鸡新城疫病毒,继续培养72 h,在倒置显微镜下观察细胞形态学变化,并测定病毒血凝效价.结果:接种病毒24 h,部分细胞产生病变;48 h,部分细胞脱落;72 h细胞单层明显破坏,培养液中病毒血凝效价可达1:512.结论:鸡新城疫病毒感染鸡胚成纤维细胞后,细胞变圆、皱缩、折光性增强,胞内形成大量合胞体,直至单层彻底破坏.     

人参皂苷及其衍生物对鸡胚成纤维细胞最大安全浓度的测定

采用组织细胞培养方法,测试了人参皂苷及其衍生物在鸡胚成纤维细胞(CEF)体外培养中的最大安全浓度。结果显示:药物在高浓度时,对CEF细胞表现出毒性,在中、低浓度时对CEF细胞具有促增殖作用。人参皂苷及其衍生物对CEF细胞的最大安全浓度为分别为375μg/mL和187.5μg/mL。     

兔胚胎体外培养液研究进展

兔胚胎体外培养研究受到广泛重视。传统的兔胚胎体外培养方法主要采用基础培养液 ,添加各种成分 ,如血清、生长因子、酶及其他营养成分促进胚胎发育。近年来 ,国内外学者对氨基酸、牛磺酸和亚牛磺酸等能源物质 ,胰岛素及胰岛素样因子 1、碱性成纤维细胞生长因子、血小板活化因子、血小板衍生生长因子和白血病抑制因子等多种生长因子 ,以及抗氧化剂如EDTA、过氧化氢酶、牛磺酸和超氧化物歧化酶等物质促进兔早期胚胎发育的作用进行了研究。此外 ,采用联合培养体系及体细胞条件培养液 ,模拟体内生长环境 ,来提供兔胚胎发育所需的物质条件也正成为研究热点     

食蟹猴-猪异种体细胞核移植的初步研究

采用食蟹猴耳部成纤维细胞作为核供体利用电融合法构建猴-猪异种核移植胚胎,研究其核重编机制并寻找适宜的培养基,初步建立食蟹猴-猪异种核移植体系。结果显示:(1)重构胚胎激活后3 h,供体核的大小基本不发生变化,但在激活后6 h大部分形成膨大的类原核。(2)食蟹猴-猪异种核移植胚胎融合率为74.2%,有70.5%的重构胚胎发生卵裂,29%的卵裂胚胎发育到桑椹胚阶段。(3)在NCSU-23+10%FCS和TCM199+10%FCS培养基中,重构胚突破4-细胞阻滞发育到8-细胞以上的比例分别为23.5%和17.0%,在NCSU-23培养基中的为19.9%,但是只有在NCSU23+10%FCS和TCM199+10%FCS培养基中重构胚胎发育到囊胚阶段(1.4%vs1.1%),尽管差异不显著。本研究首次建立了食蟹猴-猪异种体细胞克隆体系。     

Effects of Low Oxygen Tension for in vitro Oocyte Maturation and Early Embryo Development through Hypoxia-inducible Factor (HIF)

Oocytes and early embryo in vitro culture (IVC) was a key technology of mammalian embryo engineering and was the basis of biotechnology such as genetically modified (GM) and cloning research.The factors,which affect the efficiency of in vitro culture for oocytes and early embryo,include the composition,ion concentration and osmotic pressure of the culture medium,and the gas composition and temperature of the culture environment. Recent studies have shown that the oxygen tension in the culture systems can significantly affect the development of embryos,and this effect is mainly regulated by the hypoxia-inducible factor (HIF). This paper reviewed the effects of the oxygen tension on oocyte maturation and early embryo development,and the structure and regulation mechanism of HIF were discussed,in order to provide a theoretical basis for further explore the application of hypoxia method in the in vitro culture of oocytes and early embryo.     

低氧分压通过低氧诱导因子(HIF)影响卵母细胞的体外成熟和早期胚胎的发育

卵母细胞和早期胚胎的体外培养(IVC)是哺乳动物胚胎工程的一项关键技术,是生殖生物学和转基因与克隆技术等生物技术研究的基础.影响卵母细胞和早期胚胎体外培养的因素包括培养液组成成分、离子浓度和渗透压,培养环境气相组成和温度等.最近研究表明,培养系统中的氧分压可以显著影响胚胎的发育,而这种影响主要由低氧诱导因子(hypoxia-inducible factor,HIF)进行调控.作者综述了氧分压及HIF对卵母细胞和早期胚胎体外发育的影响,并对HIF的结构及调控机制进行讨论,为研究低氧培养技术在卵母细胞和早期胚胎体外培养中的应用提供依据.     

鸭胚成纤维细胞培养、传代及保存方法的研究

本研究旨在建立鸭胚成纤维细胞离体培养、传代以及冷冻保存体系,为鸭胚胎或生殖干细胞的离体培养奠定基础。利用胰酶-EDTA消化鸭胚胎组织,10% FBS+DEME营养液培养,获得原代和传代成纤维细胞。分别用DMSO、甘油以及乙二醇作为冷冻保护剂对F1代鸭胚成纤维细胞进行冷冻保存,检测复苏后细胞的生长情况。利用此方法可以获得高活性的鸭胚成纤维细胞,并可成功传至第三代。其中F1代与F2代细胞活力最好,达到90%以上。3种冷冻剂保存的细胞复苏后均与F1代细胞的活力存在明显差异,均小于80%,其中用DMSO冷冻保存的细胞复苏后活力最强,生长密度保持较好。     

In vitro production of porcine embryos: current status,future perspectives and alternative applications

The pig is considered to be a suitable source of cells and organs for xenotransplants, as well as a transgenic animal to produce specific proteins, given the biological similarities it shares with human beings. However, the in vitro embryo production system in pigs is inefficient compared with those in other mammals, such as cattle or mice. Although numerous modifications have been applied to improve the efficiency of in vitro embryo production systems in pigs, not much progress has been made to overcome the problem of polyspermy, and low developmental ability due to insufficient cytoplasmic abilities of in vitro matured oocytes and improper culture conditions for the in vitro produced embryos. Recent achievements, such as the establishment of chemically defined medium and utilization of ‘zona hardening’ technique, have gained some success. However, further research for the reduction of polyspermy and detrimental effects of the culture systems in pigs is still needed.     

Production and quality of bovine oocytes and embryos

Many factors influence the efficiency of the in vitro embryo production technology in cattle but the most important are the physiological conditions of the donor and the culture protocols for oocyte maturation and fertilization and for embryo culture from zygote to blastocyst. Therefore, general factors such as age, body conditions and herd management play a pivotal role together with more specific factors such as reproductive soundness and ovarian cyclicity. Given that good quality and competent oocytes are available a complex series of processes, including oocyte maturation, fertilization and culture of the derived zygotes, must be completed to generate viable embryos.     

A Morphological Study of Cultured Endodermal Cells of Chick Embryo: Characteristics of Adhesion, Spreading and Locomotion

A study is made of the morphological characteristics of the endodermic cells of the stage 5 chick embryo by means of in vitro cell culture techniques. The scanning electron microscope revealed that the endodermic cells in cultures were rounded, tended to be smooth and had few blebs and microvilli. Regarding cell projections typical of culture cells, such as filopodia, lamellipodia and pseudopodia, there was a noteworthy scarcity after 12 h growth, although greater cellular activity was observed at 24 h, characterized by the presence of filopodia and an ability of the cells to form clusters on the substratum. These facts show the morphological and adhesion movements of the endodermal cells studied to be related mainly with the presence of filopodia as the most abundant cell projections.     

牦牛输卵管上皮细胞和卵泡颗粒细胞的培养

分离培养牦牛输卵管上皮细胞和卵泡颗粒细胞的目的是克服体外受精中早期胚胎发育阻断,建立有利于牦牛早期胚胎体外发育的共培养体系.采集牦牛输卵管上皮细胞进行原代及传代培养,同时从卵泡液中获取颗粒细胞进行原代培养,培养过程中观察2种细胞的生长方式和形态特点.输卵管上皮细胞贴壁生长时,呈多边形,且呈单层成簇生长.培养144 h～216 h,可形成细胞单层.颗粒细胞贴壁生长时,呈现聚集生长特性,贴壁细胞形状不规则,呈放射状.培养72 h～96 h,形成颗粒细胞单层.     

Production and Quality of Bovine Oocytes and Embryos

Many factors influence the efficiency of the in vitro embryo production technology in cattle but the most important are the physiological conditions of the donor and the culture protocols for oocyte maturation and fertilization and for embryo culture from zygote to blastocyst. Therefore, general factors such as age, body conditions and herd management play a pivotal role together with more specific factors such as reproductive soundness and ovarian cyclicity. Given that good quality and competent oocytes are available a complex series of processes, including oocyte maturation, fertilization and culture of the derived zygotes, must be completed to generate viable embryos.