Proliferation and differentiation of transplantable rabbit epithelial sheets engineered with or without an amniotic membrane carrier
Proliferation and differentiation of transplantable rabbit epithelial sheets engineered with or without an amniotic membrane carrier
复制标题
DOI：
10.1167/iovs.06-0664
复制
发表时间：
2007-02-01
影响因子：
4.4
通讯作者：
Tsubota, Kazuo
中科院分区：
文献类型：
--
作者：
Higa, Kazunari;Shimmura, Shigeto;Tsubota, Kazuo
目的。报告一种使用可生物降解的纤维蛋白密封剂工程化可移植、无载体角膜上皮片的新方法，并将其特性与在裸露羊膜载体上培养的上皮片进行比较。方法。在涂有可生物降解纤维蛋白胶的培养皿中制备分层角膜上皮片。羊膜（AM）携带者作为对照。通过免疫组织化学比较培养片的细胞角蛋白（K）3、K12、K14、p63、occludin和整合素β1的质量；电子显微镜；和菌落形成测定。通过蛋白质印迹分析比较K3蛋白表达。在角膜缘缺陷兔移植模型中，通过组织学和抗 Ki67 染色检查术后适应和 BrdU 标记细胞片的增殖。通过使用可生物降解的纤维蛋白密封剂成功地设计了上皮片。两组细胞片层都是多层的，表达 K3、K12 和 K14，并且具有功能性的 occludin(+) 顶端紧密连接以及基底细胞中的 p63 和整合素 beta 1 染色。无载体片层似乎比 AM片层更具分化性，蛋白质印迹中 K3 水平较高也证明了这一点。尽管在 AM 片上观察到较大的集落，但两组中解离细胞的集落形成效率相似。移植后，与无载体片材相比，AM 片材保留了更高水平的 BrdU 标记细胞和更少的 Ki67+ 细胞。结论。使用市售纤维蛋白密封剂进行组织工程是制造无载体、可移植角膜上皮片的有效方法。与AM片层相比，无载体片层的分化程度更高，同时保留相似水平的集落形成祖细胞。
PURPOSE. To report a novel method of engineering transplantable, carrier-free corneal epithelial sheets by using a biodegradable fibrin sealant and to compare its characteristics with epithelial sheets cultivated on denuded amniotic membrane carriers.METHODS. Stratified corneal epithelial sheets were prepared in culture dishes coated with biodegradable fibrin glue. Amniotic membrane ( AM) carriers served as the control. The quality of cultivated sheets was compared by immunohistochemistry for cytokeratin ( K) 3, K12, K14, p63, occludin, and integrin beta 1; electron microscopy; and colony-forming assays. K3 protein expression was compared by Western blot analysis. In a limbal-deficient rabbit transplantation model, postoperative adaptation and proliferation of BrdU-labeled cell sheets were examined by histology and anti-Ki67 staining.RESULTS. Epithelial sheets were successfully engineered by using a biodegradable fibrin sealant. Cell sheets in both groups were multilayered, expressed K3, K12, and K14, and had functioning occludin(+) apical tight junctions as well as p63 and integrin beta 1 staining in basal cells. The carrier-free sheets appeared to be more differentiated than the AM sheets, which was also demonstrated by the higher levels of K3 in the Western blots. The colony-forming efficiency of dissociated cells was similar in both groups, although larger colonies were observed on the AM sheets. AM sheets retained higher levels of BrdU-labeled cells and fewer Ki67+ cells compared with carrier-free sheets after transplantation.CONCLUSIONS. Tissue engineering with a commercially available fibrin sealant was an effective means of creating a carrier-free, transplantable corneal epithelial sheet. Carrier-free sheets were more differentiated compared with AM sheets, while retaining similar levels of colony-forming progenitor cells.