摘要
目的:制备并保存羊膜细胞外基质,观察新鲜制备及中长期保存的羊膜细胞外基质的结构特点。方法:实验于2004-01/12在赣南医学院科研中心完成。①采用目前国际公认的羊膜制备与保存方法,制备并保存羊膜细胞外基质。②取新鲜制备的羊膜细胞外基质、中期保存的羊膜细胞外基质(4℃恒温冰箱储存备用1周)、长期保存的羊膜细胞外基质(-80℃超低温冰箱中储存备用3个月),通过大体、光镜、透射电镜和扫描电镜观察其形态及结构特点。结果:①羊膜细胞外基质大体观察:新鲜制备及中、长期保存的羊膜细胞外基质均为透明、无色、有一定韧性生物膜,其厚度约0.02～0.4mm。②光镜观察结果:新鲜制备和经中、长期保存的羊膜细胞外基质可见2层结构:基底膜厚薄不均,无细胞结构;致密层由结缔组织组成。新鲜羊膜与中期保存的羊膜细胞外基质致密层较长期保存的羊膜细胞外基质略厚;新鲜羊膜细胞外基质可见少量成纤维细胞,而中、长期保存羊膜细胞外基质的成纤维细胞较少见。③透射及扫描电镜观察结果:新鲜制备与中期保存的羊膜细胞外基质两者的基底膜厚约0.1～0.2μm,致密层厚约30～400μm,其主要结构由胶原纤维、网状纤维和基质组成,偶见成纤维细胞;长期保存的羊膜细胞外基质的基底膜厚约0.2～0.3μm,致密层厚约20～400μm,其中致密层的主要成分为胶原纤维和网状纤维,无细胞结构;三者的胶原原纤维结构完全一致。结论:羊膜经过组织工程技术处理后去除其上皮细胞或使上皮细胞失活,保留基底膜与致密层,形成羊膜细胞外基质。羊膜细胞外基质主要成分是胶原纤维和网状纤维,因其免疫原性低于羊膜并具有抗炎性,是一种特殊的细胞外基质,作为一种细胞黏附的基膜和生物支架,具有广阔的研究和应用前景。因此羊膜细胞外基质可作为体外细胞培养的生物载体而应用于基础研究。
AIM： To prepare and preserve human amniotic extracellular matrix （AECM） and investigate the structural characteristics of AECM prepared immediately and preserved for middle to long term. METHODS： The experiment had been accomplished at Center of Medical Science Research of Gannan Medical College from January to December in 2004. ①Pieces of AECM were prepared and preserved by international recognized methods as amniotic membrane. ②Immediateterm prepared AECM prepared freshly were gained, middle-term preserved AECM （preserved for a week at 4℃）, and long-term preserved AECM （preserved for three months at -80℃）. The configuration and structure characteristics of AECM were observed with sight, light microscope （LM）, transmission electron microscope （TEM） and scanning electron microscope （SEn）. RESULTS： ①AECM observed under sight： AECM prepared immediately and preserved for middle to long term respectively was a kind of lucent, colorless and definitely tough biomembrane whose thickness was about 0.02-0.4 mm. ②AECM observed under LM： There were two layer structures at AECM prepared immediately and preserved for middle to long term. One was the basal lamina that was uneven in thickness and had no cytoarchitecture; the other was the densa lamina that was composed of the connective tissue. AECM prepared and preserved for middle term were slightly thicker than that for long term. A few fibroblasts were observed with AECM prepared immediately, whereas few fibrublasts were observed with AECM prepared and preserved for middle to long term. ③AECM observed under TEM and SEM： The thickness of the basal lamina of AECM prepared immediately and preserved for middle term was 0.1-0.2 μm; the thickness of the densa lamina was about 30-400 μm, and the majority of the densa lamina were collagen fibers, reticular fibers and ground substance and the fibroblasts were observed once .in a while; The thickness of the basal lamina of AECM prepared immediately and preserved for long term was about 0.2-0.3 μm; the thickness of the densa lamina was about 20-400 μm, and the majority of the densa lamina were collagen fibers and reticular fibers, but the cytoarchitecture was not observed; The structures of the collagenous fibril were completely consistent among three different terms.CONCLUSION： The amnion is eliminated or killed its epithelia by the tissue engineering technique, and then forms AECM containing the basal lamina and the densa lamina, Because the main compositions of AECM are collagen fibers and reticular fibers and the immunogenicity of AECM is lower than that of the amnion, and AECM is a kind of special extracellular matrix. As a kind of membrane or biologic skeleton attached with cells, it has expansive research and application in the future.Therefore, AECM can be applied on the basic investigation as a biologic carrier of cell culture in vitro.
出处
《中国临床康复》
CSCD
北大核心
2006年第1期79-81,i0005,共4页
Chinese Journal of Clinical Rehabilitation
基金
江西省卫生厅资助项目(2001005)~~
