原代和传代培养的人输卵管上皮细胞对小鼠胚胎体外发育的影响

体内精卵结合、早期胚胎发育是在输卵管中进行,人们将配子、早期胚胎进行输卵管内移植（GIFT、TET）,发现胚胎发育率和妊娠率都提高。体外模仿输卵管内环境的最好方法是将胚胎与输卵管上皮细胞或组织共培养。本实验用已建立的原代和传代培养的人输卵管上皮细胞分别与小鼠早期胚胎共培养,观察其对早胚发育的影响。胚胎与不同培养时间的人输卵管壶腹部上皮细胞共培养的发育状况见表1。表1人输卵管上皮细胞对小鼠胚胎发育的影响P：原代培养第5－6d细胞;S1－S5：传代1－5代培养第4－5d细胞;T2、T4：传代第2和第4代培养第id细胞,设此…     

人输卯管组织与小鼠胚胎共培养的初步观察

为了检测人输卵管组织共培养系统对胚胎早期体外发育的影响,将超排得来的小鼠2细胞期胚胎与人输卵管组织共培养,或以人输卵管组织培养后的条件培养液进行培养。培养液为Ham's F10+10％FCS,并以其作为对照培养液。实验结果显示:无论是组织共培养或是条件培养液共培养,胚胎体外发育达囊胚率,胚胎逸出透明带率及胚胎的发育速度都较F10对照组高。其中输卵管壶腹部组织共培养及其条件培养液共培养对胚胎发育的促进作用较峡部大,而且其条件培养液共培养是以组织培养一周内所得的条件培养液效果最好。组织共培养和条件培养液共培养发育的胚胎正常分裂,没有差别,形态也均正常。实验提示人类输卵管上皮可能分泌某种因子能促进胚胎发育。     

人类输卵管上皮细胞培养

哺乳动物输卵管是生殖过程中配子输送、受精及着床前胚胎发育的场所。在体外授精一胚胎移植（IVF－ET）工程中,虽然体外培养条件已不断得到改进,但精卵结合、胚胎早期发育及受孕率仍远不及体内。有人尝试用完整的兔子输卵管体外支持牛卵体外成熟、受精及早胚发育获得成功。用羊输卵管上皮细胞与羊胚胎体外共培养,发现可促进胚胎发育并得到高活力胚胎。更进一步的研究证明输卵管上皮细胞可能通过分泌特异蛋白而对精子、卵子及早胚产生影响。目前,对该特异分泌蛋白的研究尚不完全,在体研究很受限制,所以人输卵管上皮细胞体外培养是一…     

人输卵管上皮细胞共培养系统对小鼠胚胎体外发育的影响

常规体外培养的胚胎桑椹胚以上发育率低,胚胎质量不高及移植环境不适合可能是导致IVF-ET成功率低下的原因。目前,国外一些实验室尝试将多种哺乳动物乃至人的胚胎与输卵管组织或上皮细胞进行共培养,发现有促进胚胎体外发育及提高妊娠率的作用,本课题组将人输卵管上皮细胞与小鼠胚胎共培养,观察其对胚胎体外发育及胚胎质量的影响。结果显示：共培养的胚胎24h后大部分发育到4细胞期以上,发育快的已达桑椹胚。培养48h后共培养的胚胎大部分发育成桑椹胚,有些已开始形成囊胚腔。到72h,正常发育的共培养胚胎形成囊胚和孵出囊胚。3个观察…     

胚胎单个定位及三维共培养微流控装置的构建

胚胎体外发育是临床人工辅助生殖技术中的关键环节,对着床后胚胎发育状况有重要作用。该研究利用细胞三维培养技术和微流控芯片技术构建了一种新型的小鼠胚胎体外共培养装置,用于实现自动化的小鼠胚胎定位与共培养操作,并对胚胎在体内的发育环境进行模拟。利用该微流控装置成功进行了小鼠胚胎的单个定位、三维细胞共培养以及发育追踪观察,并对装置中胚胎发育状况做出评价。统计结果表明:共培养芯片中小鼠胚胎发育囊胚率(87.3%±3.1%)显著高于对照组的囊胚率(76.8%±2.7%,P0.01);并且共培养芯片中小鼠囊胚的平均细胞总数(102±4)也显著高于对照组中的平均细胞总数(68±3,P0.01)。因此该胚胎共培养微流控芯片有助于在体外实现自动化的胚胎定位和培养过程,并且对小鼠胚胎发育具有更好的促进作用。     

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。     

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     

不同体外培养条件对牛体外受精胚胎质量影响的研究

研究探讨了不同体外培养条件下牛体外受精胚胎的发育速度及囊胚细胞数，从而了解不同体外培养系统对牛体外受精胚胎质量的影响，实验一中伴体外受精卵分别在ＳＯＦ＋牛输卵管上皮细胞（ＢＯＥＣ）和ＴＣＭ１９９＋ＢＯＥＣ两种培养系统内培养，在这两种培养条件下囊胚发育率分别为２２．２％和２１．２％，囊胚细胞数分别为１１３．３±５．０和９７．９±８．３，受精后第６～９ｄ的囊胚出现率分别为４０．１％，３７．０％，１９．     

人类输卵管上皮细胞培养

从１６例妇女的正常输卵管标本中获取壶腹部上皮细胞建立体外培养并传代，通过倒置显微镜和相差显微镜观察细胞形态及生长状况，所有标本的细胞均可在培养的６￣９ｄ形成融合的单层细胞，并可传代４￣６次，每次历时５￣７ｄ，最长的培养可维持３５ｄ，与提供标本的妇女的月经周期及年龄关系不大。     

体外培养高产奶牛胚胎的研究

研究体外培养法培养效果及对体外受精胚胎发育的影响。体外受精胚胎分别在加输卵管上皮细胞单层和颗粒细胞单层（2×106个／ml）的体外培养体系的培养液中培养。结果发现,冷冻胚胎解冻后停留时间对胚胎培养效果影响极显著（P〈0．01）;冷冻胚胎解冻后胚胎等级对胚胎培养效果影响显著（P〈0．05）,解冻后A级胚胎存活率79％、囊胚孵化率56％,显著高于B级胚胎存活率60％和囊胚孵化率39％（P〈0．05）。体外培养体系囊胚形成主要集中在第8d,加体细胞的体外培养体系显著好于不加体细胞的简单体外培养法。     

NestinCK19及PCNA在早期胚胎组织中的表达

目的：研究Nestin CK19及PCNA在胚胎早期组织器官的分布。方法：对6～10w的胚胎组织采用免疫组化SABC法染色观察。结果：胚胎6w时肝脏内血细胞前体细胞Nestin阳性；7w时肝、椎间盘及皮肤有Nestin的表达，导管上皮组织开始出SECK19阳性反应。PCNA的表达出现在肝脏，9w时表达达到高峰。结论：Nestin CK19及PCNA在胚胎早期发育过程中对肝、椎间盘、皮肤等器官组织的发育可能起着调控作用。     

静电场对小鼠胚胎发育能力的影响

首次报道哺乳动物胚胎经静电场处理后对＂细胞阻滞＂和后期发育能力的影响．结果表明，用静电场处理小鼠体外受精卵和２细胞胚胎时，对克服小鼠＂２细胞阻滞＂无显著影响．处理发育于Ｍ１６＋１００μＭＥＤＴＡ＋输卵管上皮的２细胞胚胎时，显著提高了胚胎的发育能力，最佳处理剂量的囊胚率从４２％提高到６４％，囊胚孵化率从２０％提高到４５％．     

人输卵管液和输卵管上皮细胞培养液诱发精子的顶体反应

目的：探讨人输卵管液和上皮细胞培养液对人精子顶体反应的诱发作用。方法：用Ｈａｍ′ｓＦ１０作对照组，输卵管液和上皮细胞培养液为实验组，分别与生育力组（ｎ＝２０）和不育组（ｎ＝２０）的精子进行共孵育，用精子顶体三色染色法对顶体状态进行评价。结果：共孵育３ｈ时与Ｈａｍ′ｓＦ１０对照组相比较，两种输卵管液均可显著提高生育力组和不育组的精子顶反应率（Ｐ〈０．０５），但这两种输卵管液处理的生育力组与不育组之间     

Loss of p16Ink4a with retention of p19Arf predisposes mice to tumorigenesis.

The cyclin-dependent kinase inhibitor p16INK4a can induce senescence of human cells, and its loss by deletion, mutation or epigenetic silencing is among the most frequently observed molecular lesions in human cancer. Overlapping reading frames in the INK4A/ARF gene encode p16INK4a and a distinct tumour-suppressor protein, p19ARF (ref. 3). Here we describe the generation and characterization of a p16Ink4a-specific knockout mouse that retains normal p19Arf function. Mice lacking p16Ink4a were born with the expected mendelian distribution and exhibited normal development except for thymic hyperplasia. T cells deficient in p16Ink4a exhibited enhanced mitogenic responsiveness, consistent with the established role of p16Ink4a in constraining cellular proliferation. In contrast to mouse embryo fibroblasts (MEFs) deficient in p19Arf (ref. 4), p16Ink4a-null MEFs possessed normal growth characteristics and remained susceptible to Ras-induced senescence. Compared with wild-type MEFs, p16Ink4a-null MEFs exhibited an increased rate of immortalization, although this rate was less than that observed previously for cells null for Ink4a/Arf, p19Arf or p53 (refs 4, 5). Furthermore, p16Ink4a deficiency was associated with an increased incidence of spontaneous and carcinogen-induced cancers. These data establish that p16Ink4a, along with p19Arf, functions as a tumour suppressor in mice.     

Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides

Murine embryonic stem (ES) cells are pluripotent cell lines established directly from the early embryo which can contribute differentiated progeny to all adult tissues, including the germ-cell lineage, after re-incorporation into the normal embryo. They provide both a cellular vector for the generation of transgenic animals and a useful system for the identification of polypeptide factors controlling differentiation processes in early development. In particular, medium conditioned by Buffalo rat liver cells contains a polypeptide factor, ES cell differentiation inhibitory activity (DIA), which specifically suppresses the spontaneous differentiation of ES cells in vitro, thereby permitting their growth as homogeneous stem cell populations in the absence of heterologous feeder cells. ES cell pluripotentiality, including the ability to give rise to functional gametes, is preserved after prolonged culture in Buffalo rat liver media as a source of DIA. Here, we report that purified DIA is related in structure and function to the recently identified hematopoietic regulatory factors human interleukin for DA cells and leukaemia inhibitory factor. DIA and human interleukin DA/leukaemia inhibitory factor have thus been identified as related multifunctional regulatory factors with distinct biological activities in both early embryonic and hematopoietic stem cell systems.     

Derivation of androgenetic embryonic stem cells from m-carboxycinnamic acid bishydroxamide (CBHA) treated androgenetic embryos

The androgenetic embyronic stem (aES) cells are useful models in studying the effects of imprinted genes on pluripotency maintaining and embryo development. The expression patterns of imprinted genes are significantly different between uniparental derived aES cells and zygote-derived embryonic stem (ES) cells, therefore, the imprinting related cell pluripotency needs further exploitation. Several approaches have been applied in generation of androgenetic embryos and derivation of aES cell lines. Here, we describe a method to generate androgenetic embryos by injecting two mature sperms into one enucleated oocyte. Then these androgenetic embryos were treated with a histone deacetylase inhibitor: m-carboxycinnamic acid bishydroxamide (CBHA). Further, aES cell lines were successfully derived from these treated androgenetic embryos at blastocyst stage. The CBHA could improve not only the quality of androgenetic embryos, but also the efficiencies of aES (CaES) cells derivation and chimeric mice generation. The imprinted gene expression pattern in the CBHA treated embryo-derived aES (CaES) cells was also highly similar to that of zygote-derived ES cells.     

Scatter factor is a fibroblast-derived modulator of epithelial cell mobility

Various factors are known to regulate cell growth and differentiation, but less is known of agents which affect movement and positioning, particularly in epithelial-mesenchymal interactions. Cultured human embryo fibroblasts release a protein with a relative molecular mass (Mr) of approximately 50,000 (50K) that affects epithelial cells by causing a disruption of junctions, an increase in local motility and a scattering of contiguous sheets of cells. To investigate specificity, a range of cells has been examined for the ability to produce the factor and for sensitivity to its action. Most freshly isolated normal epithelia and epithelia from cell lines of normal tissue, but not epithelia from tumour cell lines or fibroblasts, were sensitive to scatter factor. In contrast, production of the factor, as identified by activity and by chromatography, was restricted to embryonic fibroblasts and certain variants of 3T3 and BHK21 cells and their transformed derivatives. We conclude that the scatter factor is a paracrine effector of epithelial-mesenchymal interaction, which affects the intercellular connections and mobility of normal epithelial cells. The factor might be involved in epithelial migration, such as occurs in embryogenesis or wound healing.     

Human embryonic stem cell lines derived from single blastomeres

The derivation of human embryonic stem (hES) cells currently requires the destruction of ex utero embryos. A previous study in mice indicates that it might be possible to generate embryonic stem (ES) cells using a single-cell biopsy similar to that used in preimplantation genetic diagnosis (PGD), which does not interfere with the embryo's developmental potential. By growing the single blastomere overnight, the resulting cells could be used for both genetic testing and stem cell derivation without affecting the clinical outcome of the procedure. Here we report a series of ten separate experiments demonstrating that hES cells can be derived from single blastomeres. In this proof-of-principle study, multiple biopsies were taken from each embryo using micromanipulation techniques and none of the biopsied embryos were allowed to develop in culture. Nineteen ES-cell-like outgrowths and two stable hES cell lines were obtained. The latter hES cell lines maintained undifferentiated proliferation for more than eight months, and showed normal karyotype and expression of markers of pluripotency, including Oct-4, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, nanog and alkaline phosphatase. These cells retained the potential to form derivatives of all three embryonic germ layers both in vitro and in teratomas. The ability to create new stem cell lines and therapies without destroying embryos would address the ethical concerns of many, and allow the generation of matched tissue for children and siblings born from transferred PGD embryos.