CAJ | 학술논문

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.
목적 관찰완전무세포인양막기질(thoroughly acellular human amniotic membrane,TAHAM)재체외여성섬유세포적점부방식.방법 원대인포피성섬유세포(human foreskin fibroblast,HFF)충식우평명、기질효급용이매현조법제작적TAHAM.소묘전자현미경하관찰TAHAM적조직결구급초미결구.검측TAHAM적응력、응변.면역형광염색관찰Ⅰ、Ⅲ、Ⅳ、Ⅵ형효원、층점련단백、섬유결합단백、전화생장인자-β1、전화생장인자-β2、성섬유세포생장인자적표체.용도치상차현미경관찰HFF재TAHAM상적세포형태.격광공취초현미경관찰α5정합소、장단백화섬유결합단백적공간관계.결과 TAHAM내무세포,재소묘전경하TAHAM표면정섬유완정적망상결구.면역형광현시Ⅰ、Ⅲ、Ⅳ、Ⅵ형효원、층점련단백、섬유결합단백염색양성,전화생장인자-β1、전화생장인자-β2、성섬유세포생장인자염색음성.신선양막화TAHAM적과도응력、과도응변、파배응력、파배응변적차이균무통계학의의.HFF재TAHAM상정방추형,복층생장위상호련접적세포단병장입TAHAM;HFF중적α5정합소(록색)、장단백(홍색)급기분비적섬유결합단백(람색)삼색공취초형성백색조대.HFF재TAHAM상정직선운동,재평명상위무규칙운동.결론 TAHAM보류료양막적원유주요성분화역학특정,HFF가재TAHAM상형성삼유점부,기세포형태급세포이동방식부합삼유점부특정.
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.