绒山羊胚胎的体外培养,冷冻保存和移植的研究

采用体外培养和冷冻保存的方法将起排处理后的绒山羊２～８细胞期胚胎经体外在Ｍ１９９＋ＦＣＳ（对照组）、Ｍ１９９＋ＯＥＣ＋ＦＣＳ、Ｍ１９９＋ＯＥＣ＋ＥＮＧＳ和，Ｍ１９９＋ＯＥＣ＋ＮＳＳ的培养基中分别进行培养以后，选取发育为桑椹胚和早期囊胚期胚胎分别以１０％Ｇｌｙ、１０％ＥＧ为防冻剂和玻璃化方法进行冷冻保存，并将用常规和玻璃化法冷冻保存的胚胎解冻后进行移植．结果表明，体外培养胚胎的囊胚的发育率在四个培养处理组中分别为１２．５％、７０．６％、８１．８％和７３．９％，对照组与添加ＯＥＣ处理组之间具有极显著性差异（Ｐ＜０．０１），囊胚发育率在添加ＦＣＳ、ＥＮＧＳ和ＮＳＳ的实验组之间无明显差异．经冷冻保存的桑椹胚和早期囊胚的冷冻解冻胚的形态正常率在甘油、乙二醇和玻璃化三个冷冻处理组中分别为７５．０％、８６．７％和７５．０％，发育率分别为５８．３％、７８．８％和８０．０％．冷冻解冻胚的移植妊娠率在三个处理组中分别为５０％、７５％和５０％，产羔率分别为３０％、６８．８％和５０％．采用乙二醇冷冻绒山羊胚胎产生的效果优于甘油和玻璃化方法的冷冻效果。     

山羊胚胎的体外发育及冷冻保存研究

采用体外培养的方法，对山羊２～８细胞胚进行培养以后将体外发育至囊胚的胚胎进行冷冻保存，以便探讨山羊早期胚的体外培养方法和冷冻方法对体外发育胚冷冻效果的影响。将胚胎分别放入Ｍ１９９、Ｍ１９９＋输卵管上皮细胞和Ｍ１９９＋输卵管上皮细胞＋ＥＧＦ的培养液中进行培养，选取发育至囊胚期的胚胎分别以甘油和乙二醇为冷冻保护剂进行冷冻保存。结果体外培养胚的囊胚发育率在三个处理组中分别为１２．５％、７０．６％和７６．９％。使用Ｍ１９９处理组与添加输卵管上皮细胞、添加输卵管上皮细胞和ＥＧＦ的处理组之间具有极显著性差异（Ｐ＜０．０１）。冷冻解冻胚的形态正常率和发育率在以甘油和乙二醇为防冻剂的处理组中分别为６８．７、５８．３％和７８．６％、７２．８％。两组之间无明显差异，结果表明，采用与输卵管上皮细胞共同培养的方法能够提高山羊胚胎体外培养的发育率，以甘油和乙二醇为冷冻防冻剂均适合于山羊体外发育胚的冷冻保存，两者相比乙二醇的效果略优于甘油。     

不同体外培养系统对牛体外受精胚胎冷冻解冻的孵化能力的影响

采用ＳＯＦ和ＴＣＭ１９９与输卵管上皮细胞共同培养系统对牛体外受精卵进行培养,观察了在这两种培养条件下得到的襄胚经冷冻－解冻后的孵化能力,在ＳＯＦ＋输卵管上皮细胞的培养条件下,囊胚的发育率为２１．３％,襄胚经冷冻－解冻后的孵化率为３７．５％;在ＴＣＭ１９９＋输卵管上皮细胞的培养条件下,囊胚的发育率为２６．０％,衰胚经冷冻－解冻后的孵化率为４７．７％,研究结果表明这两种体外培养系统的培养效果无明显差异     

昆明鼠胚胎干细胞的分离培养与鉴定

目的：从昆明系小鼠的早期胚胎分离和培养胚胎干细胞(ES细胞)．方法：收集小鼠3．5d胚龄的囊胚，将其培养在小鼠胚胎成纤维细胞饲养层上，5—6d后取隆起生长的内细胞团块分离后再培养，观察集落的生长情况并通过碱性磷酸酶染色、原位杂交、细胞核型分析等对细胞集落进行鉴定．结果：KS细胞集落性生长，符合小鼠胚胎干细胞的一系列特性．结论：昆明系小鼠囊胚在胚胎成纤维细胞饲养层上可以发育成ES细胞，并能进行传代培养．     

非极性表面活性剂对小鼠早期胚胎冷冻保护作用的初步研究

许多研究者报道了某些非极性表面活性剂在防止蛋白质低温冷冻变性方面有着明显的效果．本研究就某些非极性表面活性剂对胚胎的防冻作用作了初步探索．方法是分别将不同浓度的两种表面活性剂Ｐｏｌｙｓｔｅｎ２０（Ｐ２０）和ＳＹ－ｇｌｙｓｔｅｒＭＬ－７５０（ＭＬ７５０）添加于改进的磷酸缓冲液（ＰＢ１）和本研究采用的防冻液（ＦＳ：ＰＢ１＋３ｍｏｌ乙烯二醇＋０．２５ｍｏｌ乳糖）中，处理小鼠早期胚２０ｍｉｎ后培养并观察对胚胎发育的影响，以确定它们对胚胎的临界安全浓度（ＣＳＣ）．在ＰＢ１中，两种表面活性剂的ＣＳＣ值均为０．５％，而在ＦＳ中，则为０．０５％．然后将两种表面活性剂Ｐ２０和ＭＬ７５０分别以０．０５％和０．０３％两种浓度添加于ＦＳ中，以ＦＳ作为对照组．在室温下平衡处理小鼠桑椹胚和早期囊胚５ｍｉｎ后连同防冻液一起装入塑料冷冻细管中．在液氮蒸气（－１７０℃）中预冷冻１ｍｉｎ后即投入液氮内保存．在３８℃水浴中快速解冻并去除防冻剂后在Ｗｈｉｔｔｅｎ培养液中培养２４ｈ．分别观察和统计冷冻－解冻胚的形态正常率和发育率．结果表明：处理组与对照组在冷冻－解冻胚形态正常率方面无显著差异（Ｐ＞０．０５），而添加０．０３％Ｐ＿２Ｏ或ＭＬ７５０的两     

小鼠囊胚的不同遗传背景对形成ES细胞集落的影响

使用正常囊胚经过内细胞团增殖后的离散程序，比较了小鼠C57BL／6品系、129品系和C57BL／6与129杂交的囊胚在形成胚胎干细胞（ES细胞）集落上的差异。C57BL／6品系的正常囊胚经培养后只有17．4％的胚胎出现ES集落，细胞生长迅速但极易分化。129品系为41．0％，细胞生长比较缓慢。而杂交鼠胚易于出现ES细胞集落，高达75．0％，有利于ES细胞系的建立。文中讨论了在嵌合体工作中使用这种杂交鼠胚ES细胞的可能性。     

牛体外受精胚胎与输卵管上皮细胞和卵丘细胞的共同培养

采用四种方法以对牛体外精胚进行培养，以便了解牛输卵上皮细胞和卵丘细胞对牛体外受精胚发育的影响。其中方法１是输卵管上皮细胞培养法，２是卵丘细胞培养法，３是输卵管上皮细胞＋卵丘细胞培养法，４是输卵管上皮＋ＥＧＦ培养法，实验１采用方法１、２和３进行培养，结果采用方法３的囊胚发育率为１９．２％，显著低于采用方法１的整胚发育率。实验２采用方法１、２和４进行培养，结果采用方法１和４的囊胚发育分别为１９．４％和     

小鼠2—细胞期胚胎细胞电融合的研究

为了探索核移植克隆动物技术中的细胞融合参数，进行了小鼠２－细胞期胚细胞的电融合试验。直流电场强度为２ｋＶ／ｃｍ，脉冲过程４０μｓ时，效果较好，可以获得７０．５％的融合率和６４．５％的发育率，进一步提高电场强度，裂解死亡率也明显提高；采用非电解质溶液作为融合液。     

兔ES样细胞系的建立及其特性分析

报道从２３７枚家兔胚胎中建成７个可连续传代的ＥＳ样细胞系。建系条件为，使用小鼠原始胚胎成纤维细胞（ＭＥ）作饲养层，以含１０％胎年血清和１０％兔血清的ＤＭＥＭ／Ｆ１２为培养基，添加白血病抑制因子（ＬＩＦ）或上皮生长因子（ＥＧＦ），胚龄为９０，９６ｈ。该细胞系的细胞。在许多方面类似于小鼠ＥＳ细胞，具干细胞的形态特征，呈集落型生长，可连续传代并保持其形态特征，具有一定的自发分化和诱导分化的能力，悬浮培养     

北京腔上囊的研究Ⅳ.胚胎后期及胚后腔上囊的发育

对北京鸭２６胚、胚后３周龄、１０周龄、１２周龄和１６周龄的腔上囊进行了比较观察，结果表明北京鸭２６日胚、胚后３周龄和１２周龄的腔上囊及其淋巴上皮滤泡发育的３个代表性阶段。２６日胚腔上囊组织结构已基本发育成熟。胚后３周龄腔上囊进入发育高峰。胚后１２周龄腔囊出现退化。综合分析光镜和电镜的观察结果，以及不同分化阶段淋巴细胞的数量统计，表明胚后期及胚后腔上囊发育过程中，分化早期的前淋巴细胞的优势所代替。     

小鼠四倍体半克隆胚胎发育研究

半克隆(Semi-Cloned)胚胎是通过注射体细胞核到未去核的卵母细胞中产生的。在半克隆胚胎中,体细胞被用来作为精子的替代物。然而,由于异常的染色体分离,构建的半克隆胚胎在激活后形成了非整倍体而导致胚胎发育受到严重影响,不能发育到期。本研究通过抑制小鼠半克隆胚胎在激活过程中染色体数目减半,避免非整倍体胚胎形成,研究四倍体半克隆(TetraploidSemi-cloned,TSC)胚胎的发育和体细胞核的掺入对胚胎发育的影响。结果显示,TSC胚胎的体外发育率显著高于二倍体半克隆胚胎,与正常受精卵及孤雌激活对照无显著性差异,但TSC胚胎的细胞数在桑椹胚和囊胚期比正常二倍体受精胚胎和孤雌激活胚胎少。通过Oct-4染色发现,TSC胚胎囊胚期内细胞团(InnerCellMass,ICM)细胞很少或者没有。移植63个四倍体半克隆胚胎到3只假孕母鼠体内,得到20个胎盘,但没有得到胎儿。组蛋白乙酰化和DNA甲基化检测显示,部分TSC胚胎在囊胚期没有形成正常受精胚胎在ICM和滋养外胚层(Trophectoderm,TE)之间的差异分布。TSC胚胎的基因表达不依赖于细胞分裂次数而依赖于发育时间。虽然TSC胚胎避免了二倍体半克隆胚胎形成非整倍体现象,但由于TSC胚胎没有ICM细胞或ICM细胞很少,所以只能形成胎盘而不能形成胎儿。本实验第一次较为全面地研究了TSC胚胎的发育,同时也为研究体细胞核再程序化、基因打靶技术提供了一种新的途径。     

纤维粘连蛋白在人滋养上皮的不同定位与功能

从正常人不同发育时期,不明原因流产,增殖型和侵蚀型葡萄胎滋养细胞角度,用免疫组织化学方法观察纤维粘连蛋白（FN）的显微定位,比较研究其不同定位与滋养上皮增殖,生长，分化,凋亡，迁移和浸润的关系。结果显示 正常人不同发育时期,FN在早孕合体滋养细胞基底膜和绒毛外滋养细胞膜呈阳性着色,在中期非合体结处的滋养细胞质呈免疫反应阳性,在足月滋养细胞呈阴性着色; 不明原因流产,FN在合体滋养细胞核内呈阳性着色; FN在增殖型葡萄胎滋养细胞膜和绒毛外滋养细胞质呈强阳性着色;FN在侵蚀型葡萄胎滋养细胞质呈阳性着色。提示FN胞质定位与滋养上皮迁移和侵蚀密切相关,FN基底膜定位与滋养细胞分化密切相关,FN胞膜定位与滋养细胞增殖相关,FN的阴性着色与滋养细胞衰老,FN胞核转位与滋养细胞凋亡可能相关。     

Regulation of mouse blastocyst adhesion,outgrowth and matrix metalloproteinase—2 by focal adhesion kinase

The interaction of extracellular matrix-integrin markedly influences the adhesion,outgrowth,differentiation and expression of serine proteinases by the blastocyst,so it is regarded as a vital factor in blastocyst implantation.Although the mechanism of extracellular interactions between extracellular matrix and integrins has been well elucidated,the roles of the signaling molecules in the extracellular matrix-integrin signal transduction pathway in blastocyst implantation are unknown.This limits the understanding of blastocyst implantation and ECM-integrin signal transduction pathway.In the present study,in vitro blastocyst culture and indirect immunocytochemistry,matrix metalloproteinases(MMPs) zymography and antisense oligodeoxynucleotide(ODN) were used to investigate the expression of a fundamental molecule of integrin-dependent signal transduction pathways,focal adhesion kinase(FAK),in mouse blastocysts and its influence on mouse blastocyst adhesion,outgrowth and MMP-2.The results showed that mouse blastocysts expressed FAK.FAK protein was clustered in the peripheral migrating trophoblast cells and dispersed in the central area of blastocyst outgrowth.Fibronectin triggered pro-MMP-2 and 64kD MMP-2 activities.The antisense ODN to FAK attnuated pro-MMP-2 and 64kD MMP-2 activites which decreased abruptly and tended to disappear with increasting concentrations of the antisense ODN.Both mouse blastocyst adhesion and outgrowth on fibronectin were also influenced by the antisense ODN.Up to 20μg/mL of the antisense ODN concentration,the adhesion and out-growth rates were decreased in a dose-dependent manner.The results indicated that FAK influenced mouse blastocyst adhesion,outgrowth and MMP-2 activity by intracellular signal transduction.In other words,FAK regulates mouse implantation in terms of blastocyst adhesive and invasive abilities.     

交联大分子的透射电镜分析

用含有不同羟值的聚乙烯醇缩要下醛与4,4′-二苯甲烷二异氰酸组成的交联聚合体系,通过分子内反应和分子间反应之间的竞争,使交联过程得以控制.合成了可溶解的分子内交联大分子.再用透射电镜对其进行观察,电镜照片显示,合成的交联大分子具有特殊结构,并显示了交联大分子的形成过程,首先形成前交联大分子(独立的球型体),然后前交联大分子再继续反应,形成分子内交联大分子.     

The expression and function of VEGF at embryo implantation “window” in the mouse

Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen that plays a critical role in angiogenesis. Recent reports indicated that VEGF was closely involved in embryo implantation and embryonic vasculogenesis. However, very little information is available about the detailed expression and function of VEGF at implantation “window”. In this work, VEGFs were primarily present on uterine epithelial cell monolayer and blastocysts including the outgrew trophoblasts at implantation window. VEGF antibodies decreased the number of mice embryos implanted and the percentage of blastocysts with attachment and outgrowth in a co-culture model in a dose-dependant manner. These findings demonstrate that VEGF is one of the essential cytokines for embryo implanta-tion in mouse. VEGF may act as a local mediator to regulate the maternal-fetal interaction, and facilitate blastocyst implantation.     

Eomesodermin is required for mouse trophoblast development and mesoderm formation

The earliest cell fate decision in the mammalian embryo separates the extra-embryonic trophoblast lineage, which forms the fetal portion of the placenta, from the embryonic cell lineages. The body plan of the embryo proper is established only later at gastrulation, when the pluripotent epiblast gives rise to the germ layers ectoderm, mesoderm and endoderm. Here we show that the T-box gene Eomesodermin performs essential functions in both trophoblast development and gastrulation. Mouse embryos lacking Eomesodermin arrest at the blastocyst stage. Mutant trophoectoderm does not differentiate into trophoblast, indicating that Eomesodermin may be required for the development of trophoblast stem cells. In the embryo proper, Eomesodermin is essential for mesoderm formation. Although the specification of the anterior-posterior axis and the initial response to mesoderm-inducing signals is intact in mutant epiblasts, the prospective mesodermal cells are not recruited into the primitive streak. Our results indicate that Eomesodermin defines a conserved molecular pathway controlling the morphogenetic movements of germ layer formation and has acquired a new function in mammals in the differentiation of trophoblast.     

Aberrant DNA methylation patterns in cultured mouse embryos

Mouse early embryos undergo genome-wide demethylation and remethylation events during pre-implantation development. Abnormal methylation reprogramming is thought to be associated with development arrest. Using immunofluorescence staining with an antibody against 5-methylcytosine (MeC), we examined the genome methylation patterns of mouse embryos cultured in vitro. The results did not show the difference in staining patterns between development-blocked two-cell embryos that cultured in vitro and the two-cell embryos that were freshly collected from the donor mice. But in vitro-arrested morulae displayed a strong positive staining when compared to the morulae freshly collected from the donor mice. At the blastocyst stage, although most embryos showed the expected methylation patterns, with highly stained inner cell mass (ICM) and weekly stained trophectoderm (TE), a proportion of embryos were dimly stained in both ICM and TE. These results indicated that the methylation profile of the embryos could be changed by culturing in vitro when the embryos were in the transition from morulae to blastocyst.     

Expression and function of a new angiogenic factor AA98 target molecule at the maternal-embryonic boundary of rhesus monkey

The target molecule of monoclonal antibody AA98 (AA for short) is a new vascular endothelial cell related factor and plays a role in angiogenesis as indicated by the previous data. To investigate its role in angiogenesis and placentation in primate, we examined its expression in the implantation sites on D17, 19, 28 and 34 of gestation in rhesus monkey by immunohistochemistry and Western immunoblot. Western blot analysis showed that the primary antibody used in this study was specific for its epitope. AA protein was mainly expressed in small blood vessels and in some cytotrophoblast cells. The AA staining was found mainly in the endothelial cells and vascular small muscle. This observation supported the AA‘s role in angiogenesis. AA was spatio-temporarily expressed in cytotrophoblasts: weak in proliferating trophoblast within cell column and endovascular trophoblast, strong in trophoblastic subpopulation within the basal plate and vascular trophoblast; AA staining within the basal plate was down-regulated during early placentation. The shift of AA98 expression in extravillous trophoblasts suggestes a role of this new factor during the course of cytotrophoblast metastasis and spiral artery remodeling. The spatio-temporarily expression indicats that AA98 could be also used as a trophoblast cellular marker to characterize the acquisition of a vascular endothelial and invasive phenotype.     

Generation of nuclear transfer-derived pluripotent ES cells from cloned Cdx2-deficient blastocysts

The derivation of embryonic stem (ES) cells by nuclear transfer holds great promise for research and therapy but involves the destruction of cloned human blastocysts. Proof of principle experiments have shown that 'customized' ES cells derived by nuclear transfer (NT-ESCs) can be used to correct immunodeficiency in mice. Importantly, the feasibility of the approach has been demonstrated recently in humans, bringing the clinical application of NT-ESCs within reach. Altered nuclear transfer (ANT) has been proposed as a variation of nuclear transfer because it would create abnormal nuclear transfer blastocysts that are inherently unable to implant into the uterus but would be capable of generating customized ES cells. To assess the experimental validity of this concept we have used nuclear transfer to derive mouse blastocysts from donor fibroblasts that carried a short hairpin RNA construct targeting Cdx2. Cloned blastocysts were morphologically abnormal, lacked functional trophoblast and failed to implant into the uterus. However, they efficiently generated pluripotent embryonic stem cells when explanted into culture.