嗅鞘细胞/细胞外基质夹层支架的制备和形态学观察

The Preparation and Morphological Observation of the Sandwich ECM-scaffold Seeded with Olfactory Ensheathing Cells

目的研究嗅粘膜嗅鞘细胞在细胞外基质夹层支架内的生长特性,为治疗神经系统损伤寻找新的移植供体。方法嗅鞘细胞/细胞外基质夹层支架由四层毯状细胞外基质支架和三层嗅粘膜嗅鞘细胞相间叠加构成。纤维蛋白原、层粘连蛋白和纤粘连蛋白按一定比例混合后,在新鲜大鼠血浆促凝作用下,构建单层毯状细胞外基质支架,在支架表面种植嗅粘膜来源的嗅鞘细胞。于倒置显微镜下观察嗅鞘细胞在支架上/内的生长过程,培养3d后,在细胞表面再次滴加上述细胞外基质混合胶以构建第二层毯状支架,依次重复两次。夹层支架制成组织切片和超薄切片后,用光镜和电镜观察和分析夹层支架的内部结构以及嗅鞘细胞在支架内的生长情况。结果由顶层至底层,支架内的嗅鞘细胞数量逐渐增多,细胞突起逐渐延长,细胞内分泌颗粒逐渐增多及其电子密度逐渐增高;细胞可在支架之间迁移,其水平排列方向具有一致性;嗅鞘细胞的细胞膜可与支架紧密粘附,支架内局部区域出现组织间隙。结论嗅粘膜嗅鞘细胞可在细胞外基质夹层支架内正常增殖和分化,细胞与支架具有良好的组织相容性。该夹层支架可作为嗅鞘细胞三维生长的载体,用于移植治疗神经损伤。

Abstract： Objective The aim of this study is to investigate the biological characteristics of the olfactory mucosa derived olfactory ensheathing cells （OECs） in the sandwich extracellular matrix （ECM） tissue engineering scaffold, and to explore a novel transplant donator for nerve regeneration. Methods The OEC-seeded sandwich scaffold was composed of four layers of mat-like ECM-scaffolds and three layers of OECs. At the role of fresh rat plasma in promoting coagulation, three matrices including fibrinogen, laminin and fibronectin each at a certain concentration constructed the monolayer ECM-scaffold on the surface of which the olfactory mucosa derived OECs were planted. Using the inverted microscope, the growth of the OECs on or in the bottom layer of the scaffold was observed, and after three days the second layer was constructed by dropping the above ECM mixture on the surface of OECs, which was repeated twice. After such sandwich scaffold was made into tissue sections and uhrathin sections, the structure of this scaffold and the OEC growth in the scaffold were observed and analyzed by light and electron microscope. Results From the top layer to the bottom one in the sandwich scaffold, the number of OECs gradually increased, cell processes extended longer and secretory granules in the cells became more with higher electron density. The OECs could migrate between the two neighboring scaffolds and arranged horizontally in the same direction. The membrane of OECs tightly adhered to the scaffold, and there was tissue space appearing in some regions of the scaffold. Conclusions The olfactory mucosa derived OECs in the sandwich ECM-scaffold underwent normal proliferation and differentiation, and had fine tissue compatibility. This kind of sandwich scaffold could serve as a novel carrier for the three dimensional growth of the OECs, which would be used for the transplanting to repair the injury nervous system.