纤维蛋白支架三维培养人羊膜细胞的生物学特性

背景：未足月胎膜早破是产科常见的妊娠期并发症，三维培养可使组织缺损恢复解剖学上的完整性，使得细胞培养成为更有实用价值的技术。 目的：通过观察人羊膜细胞在纤维蛋白支架上三维培养后的生物学特性变化，探讨细胞/支架复合物模拟体内羊膜组织用于未足月胎膜早破破口修复的可能性，并与普通平面二维培养结果进行比较。 设计、时间及地点：细胞-支架学体外观察，于2007-12/2008-11在重庆医科大学基础研究所完成。 材料：剖宫产孕妇的羊膜组织，由重庆医科大学附属第一医院妇产科提供。纤维蛋白原为Sigma公司产品。 方法：将酶消化法和反复贴壁法相结合，体外分离培养并纯化羊膜上皮细胞和羊膜间质细胞。纤维蛋白原聚合后，取处于对数生长的上皮细胞，接种于纤维蛋白支架表面，模拟胶上型三维立体培养；在纤维蛋白原聚合前，将处于对数生长的间质细胞和纤维蛋白溶液混合，模拟胶内型三维立体培养。同时将上皮细胞和间质细胞接种于24孔板中，进行普通平面二维培养。 主要观察指标：倒置显微镜及电镜下观察细胞的形态学特征，不同培养条件下间质细胞的增殖情况，间质细胞和支架的相互作用。 结果：在纤维蛋白支架表面，上皮细胞形态较平面培养圆润，细胞间可见伪足伸出，表面微绒毛丰富；在纤维蛋白胶中，间质细胞呈梭形，沿支架伸展，可见细胞在不同平面形成网络状。三维立体培养下，间质细胞增殖活性平稳，但增长幅度较平面培养缓慢。在胶内型三维立体培养中，随时间的延长纤维蛋白胶发生收缩，凝胶厚度逐渐降低，至第5天纤维蛋白胶厚度占原厚度的40%左右，第15天收缩到原厚度的10%左右。 结论：羊膜上皮细胞和羊膜间质细胞能够在纤维蛋白支架上立体生长，增殖活性平稳；在细胞-纤维蛋白支架复合物中，可观察到纤维蛋白胶随着时间推移而发生收缩的现象，提示羊膜细胞-纤维蛋白支架复合物可模拟体内组织，作为胎膜早破破口修复的生物学材料。

Biological characteristics of human amniotic cells on a three-dimensional fibrin scaffold

BACKGROUND: Preterm premature rupture of the membrane is a frequent complication during gestational period. Three-dimensional culture can help damaged tissue restore anatomical integrality, being worth for clinical application. OBJECTIVE: To explore biological characteristics of human amniotic cells three-dimensionally cultured by fibrin scaffold, study the possibility of cell/scaffold composite material for repairing preterm premature rupture of the membrane, and to compare with normal monolayer culture. DESIGN, TIME AND SETTING: An in vitro cell/scaffold study was performed at the Basic Institute of Chongqing University of Medical Science between December 2007 and November 2008. MATERIALS: Amnion tissue derived from uterine-incision delivery women was provided by the Department of Obstetrics and Gynecology, the First Affiliated Hospital, Chongqing University of Medical Sciences; fibrinogen was provided by Sigma, USA. METHODS: Either epithelial cells or mesenchymal cells, being isolated from human amniotic membrane, were cultivated using enzyme digestion and repeated adherence methods. Fibrinogen was conglomerated, and epithelial cells of logarithmic growth were plated onto the surface of fibrin scaffold to simulate a three-dimensional culture in vitro. Before conglomerating fibrinogen, mesenchymal cells of logarithmic growth were mixed with fibrin solution to simulate a three-dimensional culture in vivo. In addition, either epithelial cells or mesenchymal cells were incubated onto 24-well plate for normal monolayer culture. MAIN OUTCOME MEASURES: Cell morphology using inverted microscopy, proliferation of mesenchymal cells, and interaction between mesenchymal cells and scaffold under different culture environments. RESULTS: Epithelial cells were round and smooth on the surface of fibrin scaffold, pseudopodia were stretched out, and microvillus were rich. Mesenchymal cells in fibrin glue were fusiform in shape and stretched out along scaffold. Cells formed net structure at different surfaces. By three-dimensional culture, proliferation of mesenchymal cells was stable but slow compared to monolayer culture. By in vivo three-dimensional culture, fibrin glue gradually shrank and gel thickness gradually decreased. On the 5th day, the gel thickness was only 40% for the initial thickness, and on the 15th day, gel thickness was 10%. CONCLUSION: Both epithelial cells and mesenchymal cells can sterically grow on fibrin scaffold, and the proliferation is stable. Fibrin glue gradually shrinks in cell/fibrin composite scaffold, suggesting that amniotic cells/fibrin composite scaffold can simulate body tissues to repair preterm premature rupture of the membrane.