联合培养的成纤维细胞对平滑肌细胞增殖和形态的影响

目的:观察体外联合培养的成纤维细胞对平滑肌细胞形态和增殖的影响,为研究成纤维细胞和平滑肌细胞生物学特性和相互关系以及体外构建含此两种细胞的组织工程皮肤提供理论和实践基础。方法:在体外分别分离培养成纤维细胞和平滑肌细胞并常规传代培养。模拟组织工程皮肤的结构及成纤维细胞和平滑肌细胞间的相互影响途径,按照不同比例(平滑肌细胞:成纤维细胞=1:1,1:2,1:3)建立以胶原凝胶为基质的成纤维细胞与平滑肌细胞联合培养模型。应用荧光标记、组织学观察和MTT法对平滑肌细胞的形态和代谢进行研究。结果:联合培养模型法可以有效地建立起成纤维细胞和平滑肌细胞的相互影响模式。1:1组的成纤维细胞对联合培养的平滑肌细胞增殖有促进作用,1:3组的促增殖作用则不明显。结论:1:1联合培养的成纤维细胞对平滑肌细胞增殖有促进作用,对它们体外培养和扩增以及相互关系的研究对阐明这两种细胞组合作为真皮替代物种子细胞具有重要的意义。     

Infertility: Enhancement of outcome from intracytoplasmic sperm injection: does co-culture or assisted hatching improve implantation rates?

In two separate prospectively randomized trials, intracytoplasmicsperm injection (ICSI) cycles were studied in a controlled mannerto monitor the effects of either bovine oviductal epithelialcell co-culture (n = 119) or assisted hatching by zona drilling(n = 100). In the first study, immediately following ICSI, alleggs were placed directly either onto partial monolayers ofbovine oviductal cells or into regular culture medium. Althoughthe embryo developmental rate was apparently compromised inpart by the presence of the co-culture cells, ultimately therewere no significant differences in either the viable pregnancyrate (31.6% co-culture versus 29.0% control) or the embryonicimplantation rate (11.4% co-culture versus 13.6% control). Assistedhatching also had no significant impact on ICSI cycle outcomein terms of either the viable pregnancy rate (30.0% assistedhatching versus 32.0% control) or the embryonic implantationrate (8.5% assisted hatching versus 13.5% control). However,in female patients aged 235 years, assisted hatching appearedto convey a marginally significant benefit in terms of boththe viable pregnancy rate (35.5% assisted hatching versus 11.1%control) and the embryonic implantation rate (103% assistedhatching versus 3.1 % control). It seems that the overall improvementof ICSI cycle outcome cannot be achieved by the general applicationof either co-culture or assisted hatching. Nevertheless, itis possible that there remain specific patient groups that mightbenefit from selected use of either of these modalities.     

Fertilization and early embryology: Effects of embryo density and co-culture of unfertilized oocytes on embryonic development of in-vitro fertilized mouse embryos

We have evaluated the effects of embryo density and the co-cultureof unfertilized (degenerating) oocytes on the development ofin-vitro fertilized (IVF) mouse embryos. In experiment 1, groupsof one, five, 10 or 20 zygotes were cultured in 20 µldrops of modified human tubal fluid (HTF) medium for 168 h at38.7°C in 5% CO₂ and 95% air. As the embryo density increased,significantly (P < 0.05) higher rates of embryos reachedhatched blastocyst stage. In addition, the time required forhatching after IVF was significantly (P < 0.05) shortenedby the increase in embryo density. In experiment 2, 10 IVF zygoteswere cultured with or without 10 unfertilized (degenerating)oocytes in 20 µl drops of HTF medium. The rates of IVFembryos that developed to morula, blastocyst, expanded blastocystand hatched blastocyst stages were decreased significantly (P< 0.01) by culturing embryos with unfertilized oocytes comparedwith culturing embryos alone. In experiment 3, groups of oneor 10 IVF zygotes or 10 IVF zygotes plus 10 unfertilized oocyteswere cultured in 20 µl drops of HTF medium and the numberof cells per blastocyst was examined at 120 h after IVF. Increasingembryo density resulted in a significant (P < 0.05) increasein the number of cells per blastocyst. In contrast, the cellnumber of IVF embryos that developed to blastocyst decreasedsignificantly (P < 0.05) when they were cultured with unfertilizedoocytes. The results suggest that in-vitro development of IVFmouse embryos is enhanced by increasing embryo density and isimpaired by co-culture with unfertilized (degenerating) oocytes.     

IL-18在体外培养系统中诱导肿瘤快速杀伤效应及肿瘤特异性CTL

目的应用rhlL-18在体外培养系统(Coculture system in vitro，CCs)中诱导快速肿瘤杀伤效应及诱导肿瘤特异性细胞毒性T淋巴细胞(Cytotoxic T Lymphocyte，CTL)。方法 采用StemSep^TM免疫磁性细胞分离法分离人外周血NK细胞、T细胞及树突细胞(DCs)，流式细胞仪分析细胞表型，125I-UdR标记的细胞毒实验检测杀伤活性，ELISA方法检测IFN-7的产生量。结果 在CCs中，rhIL-18诱导出快速肿瘤杀伤效应，这种杀伤效应无抗原特异性、不受MHC限制，DCs和T细胞的存在与否对其无明显影响。在同一培养系统中，肿瘤抗原存在的条件下，96h后，rhIL-18能够诱导并促进CTL介导的肿瘤特异性杀伤效应。结论 rhIL-18能够在体外培养系统中相继诱导肿瘤快速杀伤效应及肿瘤特异性CTL。     

Ultrastructure of preimplantation human embryos co-cultured with human ampullary cells

Ova with two pronuclei were co-cultured with established human ampullary cell lines and various stages of preimplantation embryonic development were monitored by Nomarski optics and then assessed by transmission electron microscopy (TEM). Fifteen embryos ranging from the 2-cell stage to blastocyst hatching were examined for normal and abnormal features. Their ultrastructure was similar to that of embryos cultured in Whittingham's T6 medium, reported previously. Seven embryos were evidently morphologically normal and showed good organization of fine structure. Most cellular organelles underwent progressive changes during early development. There was evidence of enhanced embryonic genome activation at the 8-cell stage. Invariably, all embryos had few too many fragments, some internalized, which were later segregated into the blastocoele or found outside the trophoblast of the late morula and blastocysts. Six grossly 'normal' embryos assessed by Nomarski had multiple nuclei of various dimensions, which highlights the subjectivity of embryo assessment in the IVF laboratory. Incomplete incorporation of chromatin into nuclei and formation of micronuclei were evident in some blastomeres. The results are discussed in relation to early embryonic loss, prevalent in IVF. Significant events reported include the detection of centrioles at the 8-cell stage, cavitation of the early blastocyst and the initiation of blastocyst hatching visualized by TEM.     

小鼠囊胚与肿瘤细胞共培养时的生长行为观察

目的 观察小鼠囊胚与肿瘤细胞共培养时的生长行为。方法 应用胚胎与肿瘤细胞在体外共培养体系,观察小鼠囊胚与肿瘤细胞共培养时生长行为。结果 人肺癌细胞株A529对囊胚体外脱带、贴附及扩展的能力无显著性影响。结论 在体外小鼠囊胚同人肺癌细胞株A549间的粘附与扩展无明显的种属特异性,并与相作用的细胞的分化水平无关。     

人子宫内膜细胞共培养体系对早期鼠胚体外发育的影响

目的:研究人子宫内膜共培养体系对早期鼠胚体外发育的影响及移植后的妊娠情况。方法:将2-细胞小鼠胚胎与人子宫内膜细胞进行体外共培养,对照组为无营养细胞的单纯培养液,每日在显微镜下观察胚胎的发育情况。将培养到囊胚期的胚胎移植回小鼠的子宫腔,观察着床情况。结果:共培养体系中68.3％的2-细胞胚胎发育至桑椹胚期,50.8％发育至囊胚期,囊胚的孵化率为36.7％,胚胎的着床率为25.0％。而对照组只有24.8％的2-细胞胚胎发育至桑椹胚期,11.4％到达囊胚期,且其中大部分为早期囊胚即停止发育。另外对照组细胞碎片出现早且多,卵裂球不均匀,胚形态差,移植后胚胎的着床率仅为3.1％。结论:人子宫内膜细胞共培养体系可以促进小鼠胚胎的体外发育,改善胚胎的质量,提高着床率。     

A study on a chitosan-gelatin-hyaluronic acid scaffold as artificial skin in vitro and its tissue engineering applications

Chitosan-gelatin-hyaluronic acid scaffolds for tissue regeneration were fabricated by freezing and lyophilizing methods. The scaffolds showed a higher water uptake and retention abilities than chitosan-gelatin scaffolds did. Fibroblasts cultured in chitosan-gelatin-hyaluronic acid scaffolds grew and proliferated well, and they exhibited a strong viability. Keratinocytes were co-cultured with fibroblasts in chitosan-gelatin-hyaluronic acid scaffolds to construct an artificial bilayer skin in vitro . The artificial skin obtained was flexible and had good mechanical properties. The data from this study suggested that chitosan-gelatin-hyaluronic acid scaffolds are suitable for preparing a bilayer skin substitute.     

海马微环境对细胞分化和极性化的影响

目的 观察海马脑片微环境对不同分化程度细胞的极性和分化的影响。方法 将小鼠的海马脑片与5种不同分化程度的细胞（肝癌细胞、正常肝脏细胞、PC12细胞、正常神经细胞和骨髓间充质干细胞）进行共同培养,观察细胞的分化和极性产生情况。结果 与对照组相比,与海马脑片共培养的细胞数量明显减少。共培养的细胞在海马脑片上的分布有一定的规律,主要集中锥体细胞层和颗粒细胞层,其他地方分布均匀,类似于脑片片层化的构筑。海马微环境下低分化的细胞受海马脑片微环境的影响可以产生极性并分化,高分化的细胞不能产生极性。 结论 海马微环境对细胞的极性和分化有促进作用,对生长和分裂有抑制作用,细胞的极性变化与自身和所处的微环境有关,特别对低分化的细胞极性和分化的影响较大。