视网膜色素上皮细胞直接铺片法与剥离铺片法比较

Comparison of two methods for RPE fiat-mount preparation

目的介绍2种视网膜色素上皮（RPE）细胞铺片实验技术，比较其实验操作的难易程度及对RPE细胞形态和铺片完整性的影响。方法实验研究。直接铺片法：大鼠眼球取出后用4％多聚甲醛固定30min，解剖眼球，去除眼前段部分和眼内容物，小心剥去视网膜，将RPE贴附的眼后段部分剪成花瓣形并平整贴附在载玻片上，继续固定过夜。剥离铺片法：大鼠眼球取出后用4％多聚甲醛固定过夜，解剖眼球，去除眼前段部分和眼内容物，小心剥去视网膜，用镊子将RPE—Bruch膜复合物从眼球上分离，保存在PBS液中。结果剥离铺片法能得到完整RPE细胞铺片，RPE细胞形态完整：直接铺片法中RPE细胞部分丢失，铺片边缘的RPE细胞被部分破坏。结论直接铺片法操作简便，难度小，但容易破坏边缘的RPE细胞。剥离铺片法操作难度大，但对RPE细胞破坏程度小。

Objective This study compared two methods for preparing retinal pigment epithelium （RPE） flat mounts and identified the difficulties of each method. The influences of each method on the RPE cell morphology and the integrity of RPE flat-mount were also presented. Methods In this experimental study, in the first method, direct flat mounts were prepared from rat eyes that were removed and fixed for 30 rain in phosphate-buffered saline containing buffered 4% paraformaldehyde （PFA）. The anterior segment of each eye and retina were removed. The remaining eye cup containing the RPE was radially cut into 4 to 6 pieces from the periphery to the optic nerve head. In the second method in which the RPE was isolated, the eye was removed and fixed as described above except that fixation continued overnight. For the isolated flat-mount technique, the anterior segments and retina were removed. The RPE-Bruch membrane complex was then carefully isolated from the remaining eye cup, and the upward facing RPE flat mount was formed by several relaxing radial cuts. Results The isolated flat-mount method could be used to obtain the whole RPE flat-mount, and the morphology of the RPE cells could be maintained. However, the preparation process of the direct flat-mount method could cause loss of the RPE cells, especially those at the edge of the sclera. Conclusion The direct RPE flat-mount method is simple and easy to perform except that the RPE cells at the edge of sclera are disrupted. The isolated RPE flat-mount method is relatively more difficult, but RPE cells are well preserved.