体外着床模型中人孵化后早胚细胞角蛋白和肌动蛋白的表达

目的:建立理想的体外胚胎着床模型,并检测模型中人孵化后早胚细胞角蛋白、肌动蛋白和hCG。方法:孵化后早胚与人子宫内膜蜕膜化的基质细胞共培养,观察胚泡在基质细胞层上的定位、黏附、铺展和侵入过程;用免疫荧光染色技术,测定共培养系统中的细胞角蛋白和肌动蛋白;用免疫荧光分析技术,测定培养液中的hCG水平。结果:胚泡和基质细胞共培养5h起,胚泡开黏附在基质细胞层上,最终侵入蜕膜化的基质细胞间。共培养48h后,细胞角蛋白仅仅在滋养层细胞中表达;肌动蛋白在人蜕膜化的基质细胞和滋养层细胞中均有表达。囊胚与子宫内膜基质细胞共培养的培养液中的hCG水平明显高于囊胚单独培养的(P<0.01)。结论:成功建立了一个能反映人胚泡黏附、铺展及侵入到人子宫基质细胞的体外着床模型,细胞角蛋白、肌动蛋白和hCG在着床早胚细胞中起相应变化。

Expression of Cytokeratin and Actin in Human Hatched Blastocyst Implantation Model in Vitro

Objective: To establish an ideal human blastocysts implantation model in vitro, investigate the expression of cytokeratin, actin and hCG of human hacthed blastocysts in the model. Methods: Human hatched blastocysts were co-cultured with human endometrial decidualization stromal cell monolayer. The process of orientation, attachment, outgrowth and invasion in morphology were observed. Immunofluorescence staining for cytokeratin and actin and immunofluorescence measurement of hCG were performed. Results: Blastocysts attached to stromal cell layer after 5 h in co-culture. After 24 h in co-culture, the trophoblast protruded from two opposite poles of the blastocyst and underwent outgrowth into the stromal cell monolayer, blastocyst became bigger and invaded finally into the stromal cells. After 48 h in co-culture, cytokeratin staining was only visible in trophoblast but not stromal cells, actin staining was visible in both of trophoblast and stromal cells with distinct conformation and structure. Stromal cells had prominent linear actin filaments, aligned along the long axis of the cells. Cytokeratin staining in trophoblast cells was localized in short filaments arranged in a mesh. HCG levels in the supernate of stromal cell-blastocyst co-culture were higher than the supernate from blastocyst cultured only (P<0.01). Conclusion: An implantation model for the reflection of the process of human blastocysts attachment, outgrowth and invasion into stromal cells has been established in vitro. Cytokeratin, actin and hCG take place corresponding changes in the human implantation blastocyst cells.