羊水细胞培养污染危害及避免污染的策略

目的探讨羊水细胞培养细菌污染的实验室处理方法及避免污染的对策。方法对污染的羊水培养物及时更换培养液,并根据细菌革兰染色结果指导抗生素的应用,立即加入抗生素进行抗菌处理。同时总结自身的工作失误并查阅相关文献,建立规范的操作方法及规章制度,预防污染的发生。结果 4500例羊水标本中成功收获并报告结果4499例,因细菌污染培养失败1例。成功率为99.98%。平均收获时间7.5天。1例最终收获因核型数量未达到发报告要求,失败。结论及时、正确处理细菌污染的羊水培养标本,能挽救部分污染标本,同时建立操作规范尽量避免污染,保证实验的有序进行和结果的稳定可靠。     

人胚胎干细胞建系、培养及体外诱导分化研究现状

胚胎干细胞具有体外无限增殖和分化成三胚层细胞的潜能,它已被视为治疗多种疾病的一种新兴策略。目前胚胎干细胞常规的建系和培养技术已很成熟,并有一套国际公认的鉴定标准。但常规方法存在异源病原体污染的可能,急需研究适于标准化、无动物源性污染及可大量培养胚胎干细胞的培养体系。在现阶段,通过不同的体外诱导途径可将胚胎干细胞诱导成为胚外和三胚层来源的各种细胞,但定向分化的问题仍亟需解决。     

淫羊藿苷对猪早期胚胎体外培养一氧化氮及丙二醛水平的影响

目的 研究淫羊藿苷(ICA)单体对猪体外受精早期胚胎体外发育过程中培养液内一氧化氮(NO)和丙二醛(MDA)生成量的影响,探讨其与猪胚胎体外发育效果的关系. 方法 以NCSU-23培养液为对照组,以添加0.6mg/L淫羊藿苷为实验组(ICA组),通过硝酸还原酶法和硫代巴比妥酸法测定猪体外受精卵体外培养0～48h、48～120h、120～168h培养液滴中NO和MDA水平. 结果 ICA组(342枚受精卵)48h卵裂率(74.84% vs 63.32%)、120h桑囊率(45.08% vs 34.10%)、168h囊胚率(23.38% vs 18.04%)均极显著高于对照组(349枚受精卵) (P<0.01).在猪体外受精卵体外培养0～168h整个过程中,培养液内NO、MDA水平总体均呈上升趋势,但在胚胎发育3个阶段中ICA组平均每枚胚胎NO生成量高于对照组,MDA生成量则低于对照组 (P> 0.05). 结论 ICA有利于促进猪体外受精早期胚胎的体外发育,可能与其在一定程度上提高NO水平,并同时抑制MDA的生成有关.     

胚胎体外培养及胚胎干细胞系的建立

背景：人类胚胎干细胞体外建系成功,对人类胚胎发育机制和发育生物学研究、细胞和组织移植治疗某些疾病等领域都有重大意义。目的：综述近年来关于胚胎体外培养及建立胚胎干细胞系的研究进展,重点探讨胚胎体外培养影响因素、人废弃胚胎培养分离内细胞团建立胚胎干细胞系的方法及建立胚胎干细胞系的条件。方法：以“胚胎(embryo),胚胎干细胞(embryonic stem cell),共培养(co culture),序贯培养(sequential culture)”为检索词,由第一作者检索2000至2014年CNKI数据库和SCI数据库,获取有关胚胎体外培养、移植及胚胎干细胞建系的相关文献,并进行系统评价,最终保留58篇文献进行分析。结果与结论：胚胎体外培养条件是影响胚胎移植结局的重要因素,其中包括培养液的成分和培养体系。在过去的研究过程中,培养液的构成及应用已经发生了很大的变化,培养体系也从单一培养发展到共培养、序贯培养。伦理问题及胚胎来源的限制束缚着人胚胎干细胞系的建立,利用临床废弃的低质量的胚胎可作为建立人胚胎干细胞系的材料来源之一,有效的缓解了建立人胚胎干细胞系过程中胚胎缺乏的问题,并减少其中的伦理学纷争。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

诱导胚胎干细胞向软骨细胞分化研究进展

胚胎干细胞因其具有体外无限增殖能力和体内外分化发育的全能性,有望为细胞治疗或组织工程化组织构建提供可靠的种子细胞来源.如何诱导胚胎干细胞向目的细胞分化是目前面临的一大难题.首先回顾软骨细胞的体内发生、发育过程,继而对目前已知的能在体外培养环境中影响胚胎干细胞向软骨细胞分化的各类因素进行分析综述,并探讨进一步的研究方向.     

体外延长培养筛选低评分胚胎中具有发育潜能胚胎的可行性探讨

目的探讨了体外延长培养筛选低评分胚胎中具有发育潜能胚胎的可行性。方法将来自IVF/ICSI的第三天(D3)低评分胚胎进行体外延长培养,观察囊胚形成情况。结果307枚低评分胚胎经体外延长培养后形成53枚囊胚,囊胚率为17.26%,这些囊胚来自85例患者中的33例。结论①体外延长培养能有效筛选出D3低评分胚胎中具有发育潜力的胚胎;②D3胚胎形态评分不能完全预测其发育潜力。     

体外受精过程中胚胎污染的临床结局分析（附精液反复污染改行ICSI妊娠一例）

目的对体外受精过程中发生胚胎污染后的临床结局的探讨和分析。方法回顾性分析1997年3月至2007年12月,在本中心接受体外受精-胚胎移植（IVF-ET）治疗1634个周期中,体外受精过程中发生胚胎污染10个周期的临床结局。结果10例胚胎污染周期中移植8个周期18个胚胎,均未妊娠。其中1例精液反复污染,改行卵胞浆内单精子注射（ICSI）,移植3个胚胎,双胎妊娠。结论体外受精过程中发生胚胎污染会造成IVF-ET失败,胚胎污染的病人下一周期可改行ICSI治疗。     

胚胎小体来源早期血管新生模型的研究进展

在体胚胎的早期血管发育生物学研究由于受到胚胎体积小、细胞数量少和获取困难等技术难点而制约了其发展。胚胎干细胞的体外培养及其分化为胚胎小体等技术的建立，为早期胚胎血管发生的分子机制研究提供了可视性的研究平台。应用这些技术手段不仅可以观察血管发生早期的生理及病理变化，还使得外源性干预成为可能。本文小结了胚胎小体来源的早期成血管模型的研究，对胚胎小体血管新生分子生物学机制的进行了综述。     

恶性肿瘤细胞对小鼠2-细胞胚胎体外发育的影响

目的： 探讨生命早期形式与恶性肿瘤细胞在相同微环境下的早期胚胎发育情况和肿瘤细胞的生物学行为。方法： 建立小鼠2-细胞胚胎-恶性肿瘤细胞(HepG2,SKOV3,HNE1,Hepa1-6,B16,CHO)体外共培养模型，观察小鼠胚胎的发育状况和恶性肿瘤细胞的生物学行为。结果： 处于不同种属源性和胚层来源恶性肿瘤微环境的小鼠胚胎4-细胞形成率，桑葚胚形成率和囊胚形成率及其形成的囊胚细胞数目与对照组比较无显著差异（P>0.05）。小鼠的2-细胞胚胎能在人源性和鼠源性不同胚层来源的恶性肿瘤细胞体外培养环境中按一定的时间顺序发生卵裂、卵裂球紧密化、分化、囊胚腔形成，而肿瘤细胞的形态、增殖及核分裂相与对照组比较没有明显差异。结论： 在共培养环境中，恶性肿瘤细胞对小鼠2-细胞胚胎的发育时程没有阻滞和破坏作用，且与恶性肿瘤细胞的种属和胚层来源无关。小鼠早期胚胎能在不同种属和胚层的恶性肿瘤环境中按正常体外培养发育时程发育，这可能与早期胚胎发育的自我组织、高适应性和肿瘤细胞分泌的某些因子有关。     

人胚胎干细胞体外培养和扩增的研究进展

体外维持人胚胎干细胞未分化的增殖状态受细胞生长环境和内源性调节因子的控制。本文阐述了目前体外培养和扩增人胚胎干细胞细胞的各种系统,分析了影响细胞增殖和不分化的因素及其信号转导机制,指出了为临床细胞替代治疗疾病提供细胞来源的人胚胎干细胞培养系统的发展趋势。     

In vitro enhancement of early-stage embryos with co-culture.

Over the years, a great deal of effort has been made to maintain the viability of mammalian embryos once they have been removed from the female's uterus. Early embryo research studies used the simplest of medium (eg, physiological saline) in an effort to maintain embryo homeostasis in vitro, but little progress on embryo viability was made. The addition of heat-treated serum, buffers, and essential amino acids to culture media have shown evidence that improvements could be made in culturing mammalian embryos in vitro; however, it has become clearly evident that this was not going to be a simple task. In recent years, the most notable progress made in this research area has been made with the use of "helper" cells to co-culture early-stage embryos to hatched blastocysts. This article reviews the classic works that have contributed to the development of embryo culture systems that are now used in genetic engineering research and commercial embryo transplant units in the livestock industry. The development of trophoblastic vesicle culture methods and uterine cell co-culture systems were basic contributors to the development of the mammalian embryo co-culture systems as we know them today. Recent studies with follicular granulosa cells and oviduct epithelial cells have added much to our understanding of embryo development in vitro. Novel co-culture systems, such as culturing mammalian embryos in the amnion of a developing chick embryo, certainly offer encouragement that research efforts should continue until an optimal culture system is developed for each mammalian species. The potential uses of embryo culture systems for both humans and animals have yet to be fully understood.     

骨桥蛋白对小鼠胚泡黏附和扩展的影响及其机制

目的 探讨骨桥蛋白(OPN)对小鼠胚泡黏附、扩展的影响及机制. 方法 采用纤维蛋白铺板微滴培养法培养胚胎,观察并统计重组小鼠OPN(rmOPN)、OPN抗体及精氨酸-甘氨酸-天冬氨酸多肽(RGD)对胚泡的脱带、黏附和扩展的影响;采用24孔板培养胚胎,酶联免疫吸附测定(ELISA)法检测胚泡培养液基质金属蛋白酶2和9(MMP-2、-9)的浓度. 结果不同浓度的OPN组间胚泡的脱带率、黏附率与对照组相比无显著性差异(P>0.05);但1.0 mg/L和10.0 mg/L OPN组可促进小鼠胚泡提前扩展,72h囊胚扩展率与对照组相比差异有显著性,10.0 mg/L组72h的扩展率显著高于OPN 0.1 mg/L组(P<0.05).OPN抗体和RGD均显著抑制胚泡的脱带、黏附和扩展,与对照组相比,差异有显著性,且随着浓度的增加,抑制作用越明显.OPN促进胚泡分泌MMP-2及MMP-9,且存在时间和剂量依赖性. 结论 OPN在体外可通过促进小鼠胚泡的扩展和分泌MMP-2、-9来调节小鼠早期胚泡着床.     

Two-phase in vitro culture of explanted chick embryos

The present study describes a method of culturing chick embryos together with their surrounding area vasculosa on two different culture media in succession. Embryos in the 2nd day of incubation (stages 13, 14, 15 according to Hamburger and Hamilton, 1951) were explanted from the yolk with the aid of a ring of filter paper and transferred dorsal side up to a silicone culture dish containing the first culture medium (89.5% L-15, 10% fetal calf serum, 0.5% Antibiotics). The paper ring was clamped onto the wall of the culture dish by a steel ring so that the embryo was fixed for the culture period. After 4 +/- 1, 8 +/- 1, 12 +/- 1 hrs, the embryos were taken from the culture dishes and transferred to others containing a yolk-albumen mixture as culture medium; 81.2% of embryos survived the first phase of culture. On the second medium 50.3% of explanted embryos were still alive at stage 20 (HH), and 7.9% of them reached the 5th day of development (St 25 HH). The average length of survival in vitro was found to be influenced by both the length of the first culture phase and the stage at which embryos were explanted. This culture method may be useful for teratological tests, since in the first phase of culture, concentrations of test substances and the time of exposure can be exactly adjusted, and in the second phase, the embryo is allowed to develop quite normally, under conditions similar to those in ovo.     

Analysis of the long-term results of contact of human cell cultures with influenza virus]

Influenza A/Hong Kong/1/68 and A/Victoria/35/72 viruses may produce in primarily trypsinized human embryo kidney (HEK) cell culture a chronic form of persistence accompanied by periodic emergence of cytopathic changes arrested by additional medium change. This persistence causes morphological changes in HEK culture manifested in the substitution of epithelial elements by fibroblasts and prolongs the "life" of culture by over 100 days as compared to the controls. The chronic form of persistence of these viruses in human embryo lung diploid cell culture (HELDC) was accompanied by a cytoproliferogenic effect but did not lead to histomorphological changes and did not exert "oncogenic-lide" changes in cell membranes. Inapparent infection with A/Hong Kong/1/68 virus in HELDC did not cause any histomorphological and cytokaryological abnormalities and was accompanied by complete elimination of the virus from the culture. No oncornavirus contamination was found in HELDC cluture either in intact state or after inoculation with influenza virus.     

Cloned human embryonic stem cells for tissue repair and transplantation

One approach to overcome transplant rejection of human embryonic stem (ES) cells is to derive ES cells from nuclear transfer of the patient’s own cells. Because an efficient protocol for human somatic cell nuclear transfer (SCNT) has not been reported, several critical factors need to be determined and optimized. Our experience with domestic animals indicate that reprogramming time (the period of time between cell fusion and oocyte activation), activation method and in vitro culture conditions each play a critical role in chromatin remodeling and the developmental competence of SCNT embryos. In this review, we describe the optimization of human SCNT and derivation of human cloned ES cells. In our study, about approx 25% of human reconstructed embryos developed into blastocysts when we allowed 2 h for reprogramming to support proper embryonic development. Since sperm-mediated activation is absent in SCNT, an artificial stimulus is needed to initiate embryo development. Incubation with 10 μM calcium ionophore for 5 min followed by incubation with 2.0 μM 6-dimethyl amino purine was found to be the most efficient chemical activation protocol for SCNT using human oocytes. In order to overcome inefficiencies in embryo culture, we prepared human modified synthetic oviductal fluid with amino acids (hmSOFaa) by supplementing mSOFaa with human serum albumin and fructose instead of bovine serum albumin and glucose, respectively. Culturing human SCNT-derived embryos in G1.2 medium for the first 48 h followed by hmSOFaa medium produced more blastocysts than culturing in G1.2 medium for the first 48 h followed by culture in G2.2 medium or culturing continuously in hmSOFaa medium. The protocol described here produced cloned blastocysts at rates of 19–29%, which is comparable with the rates in cattle (approx 25%) and pigs (approx 26%) using established SCNT methods. A total of 30 SCNT-derived blastocysts were cultured, 20 inner cell masses (ICMs) were isolated by immunosurgical removal of the trophoblast, and one human cloned ES cell line (SCNT-hES1) with typical ES cell morphology and pluripotency was derived. Our approach opens the door for the use of autologous cells derived from nuclear transfer ES (ntES)-derived cells in transplantation medicine.     

Improvement of the culture conditions for the development of human preimplantation embryos

The culture of human preimplantation embryos from the 1-cell to the morula/blastocyst stage of development is not satisfactory at present. The success of various IVF laboratories ranges from 18 to 23%, therefore there is a requirement for improvement in the standard conditions used to culture the embryo. Using a limited number of 'spare' human embryos which were donated for research, in-vitro studies have been undertaken using various culture media. The results show that a significant improvement in viability is achieved using Ham's F-12 medium compared with other media presently used for culturing embryos.     

Transmission electron microscopy of mouse blastocysts activated and growth-arrested in vivo and in vitro

Summary Blastocysts from mice in a state of delayed implantation were examined by electron microscopy, either directly or after a culture period, to find out if activation and growth arrest in vitro were similar to the corresponding stages in vivo. It was found that activation in vitro, obtained by culturing the blastocysts in an outgrowth medium, was accompained by morphological signs similar to those during oestrogen activation in vivo, namely an increase in polyribosomes and glycogen granules. Analogously, growth arrest in vitro, obtained by culturing the blastocysts in a medium deprived of glucose, arginine and leucine, was accompanied by a morphology similar to that of blastocysts obtained during delay of implantation, namely scattered ribosomes of the monosome type, few membranes of endoplasmic reticulum and a lack of glycogen granules, all signs of a low metabolism. However, the morphological parallelism between the in vitro and the in vivo systems does not necessarily mean that the growth-controlling factors are the same in both cases.Activated blastocysts were transplanted to uteri of mice in delay of implantation to characterize the trophoblast ultrastructure four days after the transplantation. The experiments demonstrated that both in blastocysts activated for 24h in vivo and in those activated for 6 h in vitro the morphology reverted to that of inactive blastocysts, indicating that the functional inactivity is related to reversion into a morphologically inactive state rather than to uterine blocking of a morphologically active state.     

酶消化结合差时贴壁的方法体外分离培养人子宫内膜细胞

目的探讨酶消化结合差时贴壁培养的方法对人子宫内膜细胞进行体外培养的培养效果，以寻找一种简单高效的人子宫内膜细胞体外分离培养的方法。方法采用胶原酶消化结合差时贴壁的方法，对人正常子宫内膜细胞进行体外分离培养，并传代、冻存及复苏。光镜下观察其培养效果，通过免疫荧光法对腺上皮细胞及间质细胞进行鉴定，并分析其纯度。结果共培养人子宫内膜38份，成功培养35份，培养成功率92％，冻存后成功复苏率达97％；子宫内膜腺上皮细胞和间质细胞分别经角蛋白单抗和波形蛋白单抗免疫荧光显色为阳性，腺上皮细胞和间质细胞原代纯度均达90％以上。结论酶消化结合差时贴壁的培养方法，操作简单、培养成功率高、污染机会少、省时、维持细胞活性好，是子宫内膜细胞体外分离培养的简单高效方法。     

The dynamic structure of rabbit blastocyst coverings

Summary Under physiological conditions the zona pellucida disappears in the rabbit between Day 3 and early Day 4 post coitum (p.c.) and is replaced by a new layer, the neozona. The dissolution of the zona pellucida and the formation of the neozona was investigated in three different experimental approaches, all of them characterized by non-physiological developmental conditions for the embryo: Prevention of embryo migration from the oviduct into the uterus by postcoital (48 h p.c.) tubal ligation, in vitro culture, and asynchronous embryo transfer into uteri of recipient rabbits. Embryos of age 21/2, 3, 4 and 41/2 days p.c. were cultured for 12 to 72 h. The media used for in vitro culture were supplemented with BSA, serum or with uterine secretions that were collected either synchronously or asynchronously to the developmental stage of the cultured embryos. Three-day-old embryos were transferred into uteri of pseudopregnant foster rabbits of either synchronous (Day 3) or asynchronous stages (Day 0, 2, 4, 5, 6) and were recovered 24 to 72 h after transfer. The transformation of the coverings was evaluated by light and transmission electron microscopy. The dissolution of the zona pellucida was greatly disturbed in tube-locked embryos, and in cultured embryos if standard protein supplements (BSA or serum) had been used for in vitro culture. In many cases the zona was still completely preserved after 2 or 3 days in culture, at a time when it normally would have already been replaced by the neozona in vivo. The dissolution in vitro, however, progressed incomparably better if the culture medium had been substituted with synchronous or asynchronous uterine secretions. The formation of the neozona could not be verified in cultured blastocysts. After embryo transfer, the dissolution of the zona pellucida was completed in most cases by 2 days after transfer, irrespective of the recipients' progestational stage. Present results indicate that uterine components are essential for the dissolution of the rabbit zona pellucida. These components appear to be present in the uterine cavity constitutively, i.e. independently of the uterine progestational transformation, and need not be in synchrony with the embryo's developmental stage for dissolution of the zona. Normal formation of the neozona does not take place under the non-physiological developmental conditions of in vitro culture.