早期人胚羊膜共聚焦激光扫描光学切片及F-肌动蛋白的研究

STUDY OF OPTICAL SECTIONS AND F-ACTIN IN AMNION OF EARLY HUMAN EMBRYO WITH CONFOCAL LASER SCANNING MICROSCOPE

目的 建立羊膜组织共聚焦激光扫描显微镜光学切片的方法 ,探讨早期人胚羊膜细胞结构、分层及细胞骨架的空间结构。 方法 采用共聚焦激光扫描光学切片和荧光探针双重标记技术对妊娠 7～ 9周早期人胚羊膜进行形态观察、细胞F 肌动蛋白表达定量分析、细胞分层及厚度计算。 结果 羊膜由两层不同类型的细胞组成 ,内层细胞为多边形扁平细胞 ,外层细胞为长梭形细胞 ;内层细胞胞质中的F 肌动蛋白表达量较低 ,外层细胞胞质中的F 肌动蛋白表达量明显高于内层细胞 ,两者差异有统计学意义 ;7～ 9周人胚羊膜厚度为 30± 2 μm。其中 ,上皮层厚度为 10 μm± 2 μm ,间充质层厚度为 14μm± 2 μm。 结论 1 早期人胚羊膜由两层细胞即内层的上皮细胞层和外层的间充质细胞层组成 ,胞质中F 肌动蛋白的表达量间充质细胞高于上皮细胞 ;2 通过共聚焦激光扫描连续光学切片可以计算出羊膜各层的厚度 ;3 共聚焦激光扫描显微镜光学切片技术结合FITC 鬼笔环肽和碘化丙啶双重标记技术是观察和分析羊膜组织细胞立体结构的良好方法。

Objective To develop a method for the optical section of laser scanning confocal microscopy of amnion and to study the spatial structure of cell and actin cytoskeleton in amnion of human embryo. Methods Fresh amnion of human embryo was marked by doubling label technique using FITC-phalloidin and PI and observed morphological by confocal laser scanning microscope (CLSM) to image optical sections. F-actin expression in amniotic cells was quantified and the thickness of cell layers was also examined by CLSM. Results There are two cell layers and cell types in amnion which are the inner layer of flat polygon cells and the outer layer of elongated spindle cells,respectively.The level of F-actin expression in the cytoplasm of outer layer is apparently higher than that of inner one,and this difference shows statistical significance.The thickness of amnion of 7～9 weeks human embryo is 30±2μm.And epithelial layer is 10*!μm±2*!μm while mesenchymal layer is 14μm±2μm.Conclusion 1.Human amnion of 7-9 weeks consists of two cell layers and cell types that are the inner layer of epithelial cells and the outer layer of mesenchymal cells.The mesenchymal cells contain more F-actin in cytoplasm than that in cytoplasm of epithelial cells. 2.Analysis of serial optical sections using confocal laser scanning microscope allow measurement of the thickness of each layer of amnion. 3.Using CLSM combined FITC-phalloidin and PI double label technique to image thin optical sections is a good way to study spatial structure of amnion.;