胚胎体外培养的研究进展

胚胎质量的优劣是影响ART成功率的重要因素 ,而对其起决定作用的是胚胎体外培养条件 ,其中包括培养液的成分和培养体系。在过去的 2 5年中 ,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序贯培养、共培养和微流体培养。近年来人们对胚胎早期发育和对环境的需求以及自身的代谢特性有了不断的认识 ,该文就胚胎体外培养系统及培养液的改进进行综述。     

Ectoplacental cone induces resistance to apoptosis in high doses of interferon (IFN)-γ-treated decidual cells

PROBLEM In this study, we explored the relationship between decidual cells (DC) and interferon (IFN)-γ, in the presence or absence of ectoplacental cone (EC) using a coculture system. METHOD OF STUDY Decidual cells and EC were isolated from pregnant mice on gestation day 7.5. DCs were cultured for 48 hr and then treated with fresh EC. After characterization, they were treated with IFN-γ, and cell death was evaluated. RESULTS Interferon-γ drastically increased decidual apoptosis, which was partially reverted by the addition of EC to the IFN-γ-treated decidual culture. Moreover, the addition of EC to non-treated DC cultures was also capable of attenuating death rates. CONCLUSION Resistance to apoptosis may be induced in DC by the EC. This suggests that EC may participate in the inhibition of IFN-γ-dependent apoptosis and, therefore, play important role for DC survival in a cytokine-enriched placental environment.     

低清除率药物的代谢稳定性预测模型研究进展

药物代谢的稳定性测试是新药发现阶段的关键环节,实现药物的低清除率通常是药物代谢稳定性设计中的重要目标。如何准确评估低清除率药物的代谢稳定性参数,并用体外代谢数据预测人体药动学已经成为新药研发阶段的挑战。传统的肝微粒体模型和悬浮肝细胞模型的孵育时间短,低清除率药物无法产生足够的代谢转化,因此进一步模拟体内环境和延长肝细胞培养时间的新型模型逐渐发展起来。本文重点介绍了新型的低清除率药物代谢稳定性预测模型的原理和优缺点等,包括肝细胞传递式培养模型、单层贴壁肝细胞培养模型、共培养模型和微灌流模型等,同时对模型的发展趋势进行展望,以期为早期先导化合物的代谢稳定性检测提供借鉴和优化。     

星形胶质细胞与GnRH神经元共培养可通过谷氨酸受体介导促进GnRH神经元分泌

目的 探讨下丘脑促性腺激素释放激素(GnRH)神经元与星形胶质细胞共培养对GnRH神经元的影响及其可能的相关机制.方法 利用Transwell共培养系统,将GnRH神经元细胞株GT1-7与原代星形胶质细胞共培养10 d.免疫组织化学法检测GnRH神经元中谷氨酸受体(GluR)的分布,蛋白免疫印迹法检测GluR在GnRH神经元中的表达,并分别通过高效液相色谱法和酶联免疫吸附法检测培养上清中的谷氨酸(Glu)和GnRH的浓度变化.结果 共培养组的GnRH神经元发生mGluR5和GluR1/2/3/4从胞质到胞核的移位,在胞核中的表达较单纯GT1-7培养组显著增加(P<0.05),且Western-blot检测与免疫荧光检测结果一致.HPLC和ELISA检测结果表明,与单纯GT1-7培养组比较,共培养组上清中的Glu浓度显著减少,而GnRH的分泌则明显增加(P<0.05).结论 GnRH神经元与星形胶质细胞共培养可通过改变GluR(代谢型或离子型)在细胞内的定位与表达,从而增加GnRH的分泌.     

人肝癌细胞与成纤维细胞共育时细胞增殖率的动态变化

本文采用定位细胞培养技术，将人肝癌细胞和成纤维细胞有序地排列在同一容器内，用原位计数法分别测定两种细胞在培养１２、３６、６０、８４ｈ的细胞数，计算两种细胞在不同时相的增殖率，观察人肝癌细胞和成纤维细胞的相互影响。结果显示：肝癌细胞在１２～３６ｈ、３６～６０ｈ期间可刺激成纤维细胞的生长；在６０～８４ｈ则抑制成纤维细胞的生长。成纤维细胞在１２～３６ｈ刺激肝癌细胞的生长，在６０～８４ｈ则对肝癌细胞的生长起抑制作用。表明两种细胞之间存在着相互调控的关系     

坐骨神经外植块对脊神经节的感觉神经元高亲和力神经生长因子受体表达的影响

用细胞培养及trkA免疫组化染色方法，观察了坐骨神经外植块及巨噬细胞和雪旺氏细胞条件培养基对胚鼠脊神经节感觉神经元高亲和力神经生长因子受体表达的影响。结果显示：各组感觉神经元均产生免疫阳性反应，雪旺氏细胞条件培养基和巨噬细胞条件培养组免疫反应强度与对照组相同，联合培养组免疫反应为强阳性，与巨噬细胞条件培养组相比差别高度显著（P＜0.005）。免疫反应阳性神经元可分为2类：一类形状为圆形或椭圆形，另一类为多角形。实验组神经元和非神经元生长状况明显优于对照组。本研究结果提示：坐骨神经的溃变轴突有促进脊神经节内感觉神经元trkA表达的作用;雪旺细胞条件培养和巨噬细胞条件培养组的某些因子可直接或间接地维持感觉神经元的生存，但不促进其trkA的表达。     

Alumina particles influence the interactions of cocultured osteoblasts and macrophages.

The purpose of the current study was to evaluate the effects of alumina particles on secretion of several cytokines involved in bone resorption in cocultures of macrophages and osteoblasts. To distinguish the contribution of each individual cell type, we have established a heterologous in vitro system that makes use of mouse J774 cells and primary cultured human osteoblasts. J744 cells decreased the production of TNF-alpha when they were cocultured with osteoblasts. Treatment of J744 cells with alumina particles increased TNF-alpha secretion, but the induction was lower when cells were cocultured with osteoblasts. Secretion of IL-6 by J744 cells was very low, and increased in the presence of osteoblasts. Alumina particles were only able to stimulate the release of IL-6 by J744 cells when cells were cocultured with osteoblasts. On the other hand, incubation of osteoblasts with alumina particles enhanced the release of IL-6 and GM-CSF. Coculturing osteoblasts with J744 cells induced them to release IL-6 and GM-CSF, and treatment with alumina further increased the secretion of both mediators by osteoblasts. According to these in vitro results, it seems rather plausible that alumina particles are able to initiate an inflammatory response in vivo.     

Improved culture of individual muscle fibres with and without spinal cord explants in a collagen gel

Suspension culture of single adult rat flexor digitorum brevis (FDB) muscle fibres in Vitrogen, a purified collagen, on tissue culture plastic or glass with mesh ring supports is superior to culture upon other substrates including collagen-, laminin-, or Vitrogen-coated tissue culture plastic. The Vitrogen gel-fibre mixture which attaches to glass or plastic provides at least 10 times more fibres per dish than does plating fibres on other substrates. Use of Vitrogen gel permits variable plating densities and the production of adequate numbers of cultures for long-term experimental comparisons of acetylcholinesterase (AChE) and rhodamine-alpha-bungarotoxin (RBTX) distribution on muscle fibres. Use of 40 micrograms/ml ovotransferrin (OT) instead of chick embryo extract in the culture medium significantly improves long-term survival. Cultured fibres, with or without the addition of ventral spinal cord explants. may also be examined with electrophysiological techniques.     

人胚肺成纤维细胞与肺腺癌细胞共同培养时增殖率的变化

用一种新的细胞培养技术，选用人肺腺癌细胞及人胚肺成纤维细胞，将两者这一定顺序定位培养在同一容器内，用原位计数法分别测定两种细胞在１２，３６，６０，８４小时培养的细胞数法计算两种在不同时相的增殖率，观察人肺腺癌细胞和成纤维细胞的相互影响。     

Establishment of human tubal epithelial cells for coculture in an IVF program

Objective: Our goal was to develop a coculture system from human tubal epithelial cells for use in a human IVF program. Design: Fallopian tubes were obtained from women undergoing hysterectomy for benign disease. Ampullary epithelial cells were cultured, passaged, and frozen and the karyotype and epithelial characteristics of later passages examined. Setting: The study took place at the Assisted Conception Clinic, Walsgrave Hospital, and-the Department of Biological Sciences, University of Warwick. Results: Ampullary epithelial cells can be successfully cultured and maintained as diploid epitheliod lines until the fifth passage. Freezing does not affect subsequent culture, the cytoskeleton, or the karyotype. In excess of 12×10⁶ cells may be obtained from a pair of Fallopian tubes. Conclusions: The ease of establishing and maintaining human tubal epithelial cell cultures justifies their use in clinical IVF for human embryo cocultures, and further investigations into their mode of action should be encouraged.