In vitro and in vivo characterization of iris pigment epithelial cells cultured on amniotic membranes.
In vitro and in vivo characterization of iris pigment epithelial cells cultured on amniotic membranes.
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发表时间：
2006-08
期刊：
影响因子：
2.2
通讯作者：
K. Ohno-Matsui;K. Mori;S. Ichinose;Tetsuji Sato;Jiying Wang;N. Shimada;A. Kojima;M. Mochizuki;I. Morita
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作者：
K. Ohno-Matsui;K. Mori;S. Ichinose;Tetsuji Sato;Jiying Wang;N. Shimada;A. Kojima;M. Mochizuki;I. Morita
目的 确定人羊膜 (AM) 是否可以诱导在其上生长的人和大鼠虹膜色素上皮 (IPE) 细胞，使其在原位形成 RPE 细胞的特征，并确定在 AM 上生长的 IPE 细胞片的视网膜下移植是否可以保护营养不良的皇家外科学院 (RCS) 大鼠的感光细胞。方法将人类和 Long-Evans 大鼠的 IPE 细胞培养在经过分散酶处理的 AM 的基底膜侧。接种后两周，通过透射电子显微镜评估超微结构变化，并通过实时 PCR 和蛋白质印迹测定分化的视网膜色素上皮 (RPE) 细胞中存在的几种基因的表达水平。将在 AM 上培养的 IPE 细胞片移植到 4 周龄 RCS 大鼠的视网膜下腔中，并在移植后 12 周对眼睛进行组织学分析。结果 在 AM 上培养的 IPE 细胞显示出细胞间连接等超微结构特征，与原位 RPE 细胞类似。与在塑料平板上生长的 IPE 细胞相比，在 AM 上生长的 IPE 细胞对分化 RPE 细胞功能重要的基因（例如色素上皮衍生因子 [PEDF]、RPE65、bestropin、VEGF 和 BDNF）的表达上调更大。视网膜下移植在AM上生长的IPE细胞片后，RCS大鼠中存在的光感受器数量显着高于假注射大鼠和接受无IPE细胞的AM移植的大鼠。结论 AM 上生长的 IPE 细胞分化程度更高，表明 AM 是比塑料板更好的 IPE 细胞培养基质。当将 AM 上培养的 IPE 细胞片移植到视网膜下间隙时，RCS 大鼠的光感受器得到了更大的保护，这也支持了这一点。
PURPOSE To determine whether human amniotic membranes (AMs) can induce human and rat iris pigment epithelial (IPE) cells grown on them to develop characteristics of RPE cells in situ better than IPE cells grown on plastic plates, and to determine whether subretinal transplantation of IPE cell sheets grown on AMs can protect photoreceptor cells in dystrophic Royal College of Surgeons (RCS) rats. METHODS IPE cells from humans and Long-Evans rats were cultured on the basement membrane side of dispase-treated AMs. Two weeks after seeding, ultrastructural changes were evaluated by transmission electron microscopy, and the level of expression of several genes present in differentiated retinal pigment epithelial (RPE) cells was determined by real time PCR and western blotting. IPE cell sheets cultured on AMs were transplanted into the subretinal space of 4-week-old RCS rats, and eyes were analyzed histologically 12 weeks after grafting. RESULTS IPE cells cultured on AMs showed ultrastructural features like intercellular junctions, similar to RPE cells in situ. IPE cells grown on AMs had a greater upregulation in the expression of genes important for the function of differentiated RPE cells (e.g., pigment epithelium-derived factor [PEDF], RPE65, bestrophin, VEGF, and BDNF) than IPE cells grown on plastic plates. The number of photoreceptors present in RCS rats after subretinal transplantation of IPE cell sheets grown on AMs was significantly higher than that of sham injected rats and rats receiving transplantation of AMs without IPE cells. CONCLUSIONS The more advanced degree of differentiation of IPE cells grown on AMs indicates that AMs are a better substrate to culture IPE cells than plastic plates. This was supported by the greater protection of photoreceptors of RCS rats when IPE cell sheets cultured on AMs were transplanted in the subretinal space.