INTRODUCTION
Müller cells and retinal pigment epithelium cells (RPE) play very important roles in the maintenance of visual function, and they are involved in many pathological processes. For example, experiments conducted to examine the effects of proliferative vitreoretinopathy (PVR) showed that the proliferation of Müller cells and RPE resulted in the formation of proliferative membrane. In the present study, Müller cells were cocultured with RPE both under normoxic and hypoxic conditions to observe the effect of RPE on the proliferation and migration of Müller cells.
MATERIALS AND METHODS
Cell Culture Newborn pigmented rabbits (810 days old, provided by Experimental Center of Harbin Medical University) were used for primary culture. Five rabbits were anesthetized and sacrificed, after which the eyeballs were aseptically removed.
Müller cells culture The eyeballs were dissected along the ora serrata. Lens and vitreous body were removed. Then the pigment epithelium was removed from the neurosensory retina, and the latter was cut into small pieces. The retinal tissue was cultured in a dish with 5mL Dulbeccos Modified Eagle Medium (DMEM), supplemented with 200mL/L fetal bovine serum (FBS), at 37℃with 50mL/L CO2. About 7 days later, the tissue block adhered to the wall, and cells began to migrate out of the margin of the block. The obtained cells were cultured as previously described [1]; subsequently they were immunocytochemically identified by two specific markers, GFAP antibody and S100 antibody. The secondto fourthpassage RPE cells were used in these studies.
RPE culture The anterior segment and lens were separated. Afterwards, the neural retina and any remaining vitreous body were removed. The remaining optic cup was washed with phosphatebuffered saline (PBS), and a solution of trypsin (2.5g/L) was applied to the cup for 30 minutes at 37℃. The cells were then gently scraped off the posterior layer of the optic cup into the trypsin solution, and an equal volume of DMEM supplemented with 100mL/L FBS was added to neutralize enzyme activity. The final cell suspension was transferred to a 10mL centrifuge tube and spun at 1000 rpm for 5 minutes. The supernatant was discarded and the cells were resuspended in DMEM, supplemented with 100mL/L FBS, then transferred into culture dishes at 37℃ with 50mL/L CO2 [2]. The homogeneity of cultured RPE cells was confirmed by mAb to cytokeratins. The second generation cells were used for experiment.
Assay for Proliferation of Müller Cells Two assays were performed to observe the proliferation of Müller cells. One detected the change of Müller cells at various time points; another detected the change of Müller cells under various conditions. Hypoxia was achieved using CoCl2 at a concentration of 200μmol/L. Assay for various time points Transwell system (0.4μm) was used to develop a coculture system[3]. In the coculture group, the RPE (200μL, 5×104/mL) were cultured in the upper wells (insert), while the Müller cells (500μL, 5×104/mL) were cultured in the lower wells. As controls, there were no cells cultured in the lower wells[4]. In both the coculture group and control group, Müller cells were measured by MTT at various time points (3, 6, 24 and 48 hours). Table 1 A values of the control group and the coculture group at various time points(略)
Absorption was measured by a scanning multiwell spectrophotometer at 570nm, recorded as A value. There were 5 separate experiments, each performed in duplicate at each time point; therefore, 20 wells were used for each group.
Assay for various conditions Transwell system (0.4μm) was used to develop a coculture system. In the coculture group, the RPE cells (200μL, 5×104/mL) were cultured in the upper wells (insert), while the Müller cells (500μL, 5×104/mL) were cultured in the lower wells. There were no cells cultured in the upper wells in the Müller only group. The proliferation of Müller cells was measured by MTT under various conditions at 24 hours. There were 5 separate experiments carried out in duplicate under different conditions.
Assay for Migration of Müller Cells Two assays were performed to observe the migration of Müller cells. One was conducted to detect the change of Müller cells at various time points, and the other was conducted to observe any change of Müller cells under various conditions. Hypoxia was achieved by use of CoCl2, with a concentration of 200μmol/L.
Assay for various time points Transwell system (8μm) was used to develop a coculture system. In the coculture group, the Müller cells (200μL, 5×104/mL) were cultured in the upper wells (insert), while the RPE cells (500μL, 5×104/mL) were cultured in the lower wells. As controls, there were no cells cultured in the lower wells. The average number of cells under three observed fields was calculated under a phasecontrast microscope ×400. There were 5 separate experiments carried out in duplicate at each time point; therefore, there were 20 wells for each group.
Assay for various conditions Transwell system (8μm) was used to develop a coculture system. In the coculture group, the Müller cells (200μL, 5×104/mL) were cultured in the upper wells (insert), while the RPE cells (500μL, 5×104/mL) were cultured in the lower wells. There were no cells cultured in the lower wells in the Müller only group. The number of cells which migrated through micropores and stayed on the outer bottom side of the insert system were calculated at 24 hours under a phasecontrast microscope ×400. In addition, the average number of cells under three observed fields was calculated under a phasecontrast microscope ×400. A total of 5 separate experiments were carried out in duplicate under different conditions.
Data Processing and Analysis Statistical analysis was performed using SPSS13.0 statistical software. Repeated ANOVA was applied to data collected at various time points. Table 2 A values of Müller cell proliferation in factorial design study(略)
Time was treated as the withinsubject factor and the group as the betweensubject factor. Factorial experiments were employed to analyze the data of various conditions. The level of significance was set at P=0.05.
RESULTS
Cell Culture On Day 7 of primary culture, the tissue blocks were observed to be sticking to the wall. Several pseudopodiums had developed and gradually crept out of the tissue block to form a single cell. On Day 14, more than 80% fusion cells were found. More than 95% subcultured cells showed positive staining results using GFAP or S100 immunohistochemistry. Under transmission electron microscope (TEM), cells presented enriched cytoplasm, large nuclei and 810nm characteristic midsilks. RPE cells displayed intense pigmentation and presented cobblestone morphology. More than 95% of the cells showed positive staining by cytokeratin immunocytochemistry. On the basis of morphologic appearance and the expression of yellowbrown through cytokeratin immunocytochemistry, the RPE cells were confirmed.
Proliferation of Müller Cells Proliferation of Müller cells in the control group and the coculture group was detected by MTT at various time points. The MTT results are shown by A values in Table 1. Neither the difference between 3 and 6 hours nor between 24 and 48 hours was statistically significant. Nevertheless, there was statistically significant difference among other time points. Statistically significant difference between the control group and the coculture group was found.
A 2×2 factorial design study (condition [CO]: normoxia, hypoxia; culture [CU]: Müller cells and RPE, Müller cells only) showed that the main effects both of CO and CU were significant for proliferation of Müller cells, whereas the interaction term (CO×CU) was not significant. The MTT results under different culture conditions are shown by A values in Table 2 and Figure 1.
Migration of Müller Cells The number of Müller cells migrating through micropores and staying on the outer bottom side of insert systems at different time points are given in. Statistically significant difference was found among four different time points, except between 3 and 6 hours time points. There was statistically significant difference between the control group and the coculture group.Table 3 Number of Müller cells migrating in the control group and the coculture group atvarious time points(略)Table 4 Migration of Müller cells in a factorial design(略)
A 2×2 factorial design study (culture [CU]: Müller cells and RPE, Müller cells alone; condition [CO]: normoxia, hypoxia) showed that the main effects both of CU and CO were significant for migration of Müller cells, whereas the interaction term (CU×CO) was not significant. Table 4 and Figure 2 showed the migration of Müller cells in the factorial design.
[1] [2] 下一页 |