培养人视网膜色素上皮细胞脂褐素形成对胞内钙离子和细胞活力的影响.docxVIP

培养人视网膜色素上皮细胞脂褐素 形成对胞内钙离子和细胞活力的影 响 【摘要】 目的：观察培养人视网 膜色素上皮(retinal pigment epithelial , RPE#田胞在吞噬牛光感受器外节 (photoreceptor outer segments , POS膜 盘形成脂褐素的过程中， 胞内钙离子浓度及 细胞活力的变化。 方法：将培养人RPEffl 胞与制备的2X 1010 /L牛POS^培养，在 形成脂褐素过程中，用钙荧光指示剂 Fluo-3/AM和激光扫描共聚焦显微镜 (laser scanning confocal microscope , LSCM)观察培养，1, 2, 4和8d时间点胞内 钙离子的变化；用MTT比色法观察培养，1, 2, 4和8d的细胞活力。结果：对照组 RPE 细胞内钙离子荧光强度为土， 而与POS?养 的细胞内钙离子荧光强度在达到高峰值 土 (P ), 4d 下降至土(P )，但至8d仍维持 在较高值土 (P )。细胞内脂褐素自发荧光 平均强度在培养8d达到土 (P ),而对照组 为土。MTT法显示共培养，1 , 2, 4和8d细 胞相对增殖率分别为 ％ % %唏日％细胞 增殖活力在共培养1d达到最大值，之后下 降。结论：在培养人 RP#田胞和牛PO洪培 养形成脂褐素过程中，钙离子大量内流，细 胞增殖活力增加。 【关键词】脂褐素视网膜色素上皮细胞 钙离子活力 MTT光感受器外节 Effects of the formation of lipofuscin on the calcium and the activity of cultured human retinal pigment epithelial cells Abstract AIM: To observe the calcium flux and the activity of the cultured human retinal pigment epithelial (RPE) cells in the formation of lipofuscin which results from RPEphagocytizing the membranous discs shed by bovine photoreceptor outer segments (POS). METHODS: The cultured human RPE cells were fed with 2 x 1010 /L bovine POS. During the formation of the lipofuscin, the cells were analyzed with the fluoresence Ca2+ dye Fluo-3/AM and a laser scanning confocal microscope (LSCM). The activity of cells was measured with tetrazolium salt (MTT) assay for 12 hours, 1,2, 4 and 8 : Calcium fluorescence intensity (CFI) of normal RPEcells was 士 But in cells were fed with POS, it reached the peak with 士 (P ) for 12 hours and decreased to 士 (P ) for 4 days, then maintained a high level of 士 for 8 days. The mean autofluoresence intensity of the lipofuscin in RPE cells was 士 (P ) for 8 d compared to 士 in controls. MTTassay showed that the proliferation rate of the cells fed with POS were %, %, %, % and % for 12 hours, 1,2, 4 and 8 days, respectively. The activity of RPEcells showed its highest level for 1 day and then declined. CONCLUSION: In the formation of the lipofuscin when RPE phagocytizing bovine POS, calcium influx to RPEcells mayplay an