利用牛胶原构建人工真皮

The study on how to make the artificial dermis by bovine collagen gel

目的制备成纤维细胞胶原凝胶(即真皮替代物),以研究细胞的生长、分化、增生及真表皮间的相互作用,为研制大面积创面覆盖物-复合皮奠定基础。方法(1)酶消化法培养胎儿成纤维细胞;(2)制备细胞胶原凝胶,分为Ca-gel和Cf-gel组,接种后于不同时间观察两组细胞的形态变化和胶原凝胶的收缩情况;(3)细胞计数;(4)SEM观测胶原凝胶的表面是否有成纤维细胞附着;(5)HE染色。结果(1)成纤维细胞形态及增殖情况:成纤维细胞胶原凝胶形成后呈半透明状,可出现较明显的细胞增殖,并可见凝胶收缩现象,但Ca-gel组较小;人工真皮质地与颜色近似真皮组织,有一定的弹性和抗拉性,不易脆。(2)细胞计数法所得两组生长曲线基本相似。Ca-gel和Cf-gel组细胞均具有典型的细胞增殖现象,Cf-gel组细胞在后期的增殖略快于Ca-gel组。(3)扫描显微镜下可见到凝胶表面有梭形的成纤维细胞出现,且成纤维细胞紧密贴附于凝胶上。(4)HE染色可见胶原呈较均一的淡红色网状分布,成纤维细胞分布于胶原凝胶内部和表面。结论(1)培养好的细胞凝胶称之为真皮替代物,是进行皮肤器官型培养和制作复合皮的关键与基础,并可与培养的人工表皮膜片混合移植,起到促进表皮细胞的生长粘附和加速创面愈合的作用。(2)成纤维细胞在胶原凝胶中有着活跃的生物学特性。

Aim To transplant the fibroblast as the seeding cell into the bovine collagen gel in order to investigate the growth, differentiation, proliferation and function between the epidermis and dermis, simultaneously, as a basis to prepare the artificial composite skin.Methods (1)Trypsin was used as the isolation medium to get the fibroblast; (2)To prepare fibroblast collagen gel. The artificial skins were divided into two groups: Ca gel group and Cf gel group, in which the change of cell configuration and the shrinkage of gel were observed.(3)To count the cell number;(4)Whether the cells attached into the gel was observed by Scanning Electron Microscope(SEM);5. H.E stain.Results (1)Congfigration and Proliferation of fibroblasts: the fibroblast collagen gel was translucency; proliferation of the cells could be clearly observed, but the gel contracted several days after, the dermis in Ca gel group contracted less significantly than that in the Cf gel group;the character and color of the artificial dermis were similar to the normal dermis and the artificial dermis was not fragile. (2)The growth curve of two groups was similar. Cell proliferation was significant in two groups, and the cells in Cf gel group grew a little faster than those in Ca gel group during the anaphase.(3)There were fusiform cells on the surface of the gel by the scanning microscope, and the cells tightly adhered to the collagen gel. (4) The collagen gel showed even damask reticular distribution stained by H.E, the fibroblasts distributed on the surface and in the gel.Conclusion (1)The cultured cell gel was named as dermis institute,which was the key to provide the artificial composite skin, and it could be transplanted to the wound with the cultured artificial epidermis, which could facilitate the keratinocytes adhere to the dermis and wound healing quickly.(2)Based on the active biological chapters of the fibroblasts, the three dimension culture system made by the collagen gel was more scientific than the cell matrix system in the research of the tissue engineering of skin and biological behavior in fibroblasts.