| OBJECTIVES:1. To establish the method of culturing human retinal capillary endothelial cell in vitro.2. To detect the effect of glucose of high concentration on the barrier function of cultured human retinal capillary endothelial cell, and to discuss the mechanism in it.METHODS:1. Take the normal human retinas from eye bank, through washing, cutting, digesting by type I collagenase and filtering by 75μm and 50μm cell meshes, and then incubating with CD31 IgG labelled immunomagnetic beads, isolating by magnetic cell isolation system, to get the purified human retinal capillary endothelial cells.2. Identify the cells of serial subcultivation by immunohistochemistry and FCM using mouse anti-human Von Willebrand factor antibody and FITC labelled goat anti-mouse IgG antibody.3. Passage the cells and divide to 3 groups in random according to the glucose concentration: low glucose, middle glucose and high glucose, andchange the media of the cells to DMEM containing glucose of 5.6mmol/L, 15.6mmol/L and 25mmol/L respectively when the cells proliferate confluently.4. On the 3rd day and 1, 2, 3 and 4 weeks after changed the media, survey the endothelial cell activity of each group by MTT;measure TER of each group by cell resistometer;detect the express of tight junction protein Occludin and ZO-1 by western blot;and detect the gene transcription of Occludin and ZO-1 by RT-PCR.RESULTS:1. The human retinal capillary endothelial cells of primary generation and serial subcultivation could form stable monolayer cell like pavestone shape.2. The percentage of human retinal capillary endothelial cells in whole cells was more than 95% by immunohistochemistry, and it was 95.92% by FCM.3. The differences of cell activity were not significant statistically among the 3 groups at each time point.4. TER ascended gradually after the cells subcultured to Millicell HA 0.45|a.m culture plate insert, and get peak value of 29.83±2.49:Q-cm2. After change to media containing glucose of different concentration, TER fluctuated between 27.25±4.22￡"2-cm2~30.00±2.98Q-cm2 and thedifferences were not significant statistically in low glucose group;TER descended in middle glucose group, which descended rapidly between 1 to 3 weeks especially;in high glucose group, TER descended to 15.75±3.30Q-cm2 rapidly on the 3rd day, and the difference was significant statistically compared to start, and descended continuously thenceforth, but the differences were not significant statistically any longer.5. In western blot test, the express of Occludin and ZO-1 descended along with the concentration of glucose ascending at each time point. In low glucose group, the express of Occludin didn't change along with the time, but in middle and high glucose groups, the express of Occludin descended evidently along with the time;the change of the express of ZO-1 along with the time was not obvious in each group.6. In RT-PCR test, the gene transcription of Occludin descended along with the concentration of glucose ascending at each time point, but the transcription of ZO-1 was not correlation with the concentration of glucose. Along with the time, the gene transcription of Occludin changed unconspicuously in each group, but the gene transcription of ZO-1 descended conspicuously in high glucose group and unconspicuously in middle and low glucose group.CONCLUSIONS:1. The human retinal endothelial cells could be purified, cultured andsubcultivated successfully.2. The activity of human retinal endothelial cells could not be decreased by glucose of high concentration, but TER of them could be decreased along with concentration of glucose accenting. It maybe related with the gene transcription and protein express of Occludin and ZO-1.
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