组织工程构建视网膜色素上皮细胞膜片移植后结构重建及功能评价

项目名称： 组织工程构建视网膜色素上皮细胞膜片移植后结构重建及功能评价

项目编号： No.30871315

项目类型： 面上项目

立项/批准年度： 2009

项目学科： 化学工业

项目作者： 洪晶

作者单位： 北京大学

项目金额： 29万元

中文摘要： 视网膜色素上皮细胞（RPE）完整的单层和正常极向排列对其行使清除视觉细胞脱落的膜盘碎屑，并且分隔高血管的脉络膜和光感受器的屏障功能都非常重要。其功能障碍会导致严重视网膜疾病。目前RPE移植是治疗此病的有效方法。RPE细胞移植因分散植入不能建立有效连接和异体RPE细胞移植存在排斥反应的问题，限制了这项手术的开展。为此，实现永久替代细胞移植就成了亟待解决的问题。本课题将大鼠骨髓间充质干细胞提取、鉴定后在体外诱导分化，应用免疫组化、PCR、Western blot等方法比较MSC诱导前后与自体RPE细胞中Mertk基因的表达，结合特异及非特异性吞噬结果，首次证明MSC表达Mertk基因，并具有特异性吞噬ROS的功能，与自体RPE细胞比较无显著性差异；将MSC制备成无支架复层细胞膜片，体外检测具备吞噬功能及细胞间连接；首次采用经巩膜外路视网膜下移植模型将MSC无支架复层细胞膜片移植于视网膜下腔。体内观察1个月后FFA检测证明移植细胞膜片具备屏障功能，冰冻切片检测Mertk、ZO-1、Occludin的表达阳性，从而证明该细胞膜片具备一定的特异性吞噬和屏障功能，可以行使保护视网膜神经层的作用。

中文关键词： 骨髓间充质干细胞;特异性吞噬;视网膜下腔移植;结构重建

英文摘要： As a part of the visual process, it is very important for the retinal pigment epithelium (RPE) cells with a complete monolayer and normal pole to the arrangement of its exercise of the normal physiological functions. As a part of the retinal barrier, it is an important barrier to separate high vascular choroid and photoreceptors. They have to keep clear the disc debris of visual cell membrane. Its dysfunction can cause serious disease of the retina. RPE cell transplantation is effective method to treat the disease, but it is difficult to obtain autologous RPE cells and dispersion of implanted RPE cells can not establish a valid connection. Because of the rejection problems of allogeneic RPE cells, the surgery was limitated. The study is to use MSC of BN rats which was cultured and identified in vitro. With immunohistochemistry, PCR and Western blot analysis, comparing the induced MSC and MSC with autologous RPE cells. Combined with the three kinds of cells specificity of different time points (Rod the Outer the Segment ROS) and non-specific phagocytosis (phagocytosis of latex microspheres) results, it was concluded that there was no significant difference between MSC and RPE cells in phagocytosis. Then the cultured MSC was prepared to a stentless stratified cell patch, and in vitro phagocytosis and the detection of connections between cells was made. A stentless stratified MSC membrane graft was transplanted through scleral into the subretinal space. FFA was used to detect the barrier function of the transplanted patch and the expression of Mertk、Occludin、ZO-1 and Rhodopsin were positive. In conclusion, the cell patch has a certain degree of phagocytosis and barrier function.

英文关键词： Bone-Marrow Mesenchymal Stem Cells; phagocytosis; transplantation into subretinal space; Structural remodeling