成纤维细胞与完全脱细胞人羊膜的三维黏附

摘要

目的 观察完全无细胞人羊膜基质(thoroughly acellular human amniotic membrane,TAHAM)在体外与成纤维细胞的黏附方式.方法 原代人包皮成纤维细胞(human foreskin fibroblast,HFF)种植于平皿、基质胶及用胰酶悬吊法制作的TAHAM.扫描电子显微镜下观察TAHAM的组织结构及超微结构.检测TAHAM的应力、应变.免疫荧光染色观察Ⅰ、Ⅲ、Ⅳ、Ⅵ型胶原、层黏连蛋白、纤维结合蛋白、转化生长因子-β1、转化生长因子-β2、成纤维细胞生长因子的表达.用倒置相差显微镜观察HFF在TAHAM上的细胞形态.激光共聚焦显微镜观察α5整合素、桩蛋白和纤维结合蛋白的空间关系.结果 TAHAM内无细胞,在扫描电镜下TAHAM表面呈纤维完整的网状结构.免疫荧光显示Ⅰ、Ⅲ、Ⅳ、Ⅵ型胶原、层黏连蛋白、纤维结合蛋白染色阳性,转化生长因子-β1、转化生长因子-β2、成纤维细胞生长因子染色阴性.新鲜羊膜和TAHAM的过渡应力、过渡应变、破坏应力、破坏应变的差异均无统计学意义.HFF在TAHAM上呈纺锤形,复层生长为相互连接的细胞团并长入TAHAM;HFF中的α5整合素(绿色)、桩蛋白(红色)及其分泌的纤维结合蛋白(蓝色)三色共聚焦形成白色条带.HFF在TAHAM上呈直线运动,在平皿上为无规则运动.结论 TAHAM保留了羊膜的原有主要成分和力学特征,HFF可在TAHAM上形成三维黏附,其细胞形态及细胞移动方式符合三维黏附特征.%Objective Three-dimension (3D) cell matrix adhesion in vivo is fundamentally important for a wide variety of cellular physiological and pathological phenomena, however, the cell-matrix 3D adhesion is hardly observed in vitro. We present the human foreskin fibroblasts (HFF) formed 3D adhesion complexes on the thoroughly acellular human amniotic matrix (TAHAM). Methods TAHAM were produced by suspending digestion with trypsin. The HFF were seeded on 6 well plate, matrigel and TAHAM individually.The light microscope, scanning electronic microscope, immunohistochemistry and immunofluorescence were used to observe the micro-structures and detect the type Ⅰ , Ⅲ, Ⅳ, Ⅵ collagen, laminin, fibronectin, TGF-β1, TGF-β2, FGF of the TAHAM. Phase contrast microscope was engaged to observe the morphology of HFF. The time-lapse CCD and the trace analysis software were employed to prescribe the cell migration. The 3D adhesion foci were identified by the laser confocal microscope. The strain of the TAHAM was tested by the universal mechanical testing instrument. Results The fibers of the TAHAM were intact, type Ⅰ , Ⅲ,Ⅳ, Ⅵ collagen, laminin, fibronectin were positive, TGF-β1, TGF-β2, FGF were negative. HFF had a bipolar extension to form multilayer cell clusters networks and grew into the matrix. All of the seeded cells survived three weeks under regular culture without transfer. On TAHAM, HFF moved in a straight line with a speed of 12 μm/h. α5 integrin (green), paxillin (red) and fibronectin (blue) co localized to form 3D adhesion complexes (white). Conclusion The main molecular components and biomechanical properties is preserved in TAHAM. HFF forms 3D adhesion complexes on TAHAM. Cell morphology and migration of HFF on TAHAM correspond to that under 3D adhesion behavior.