¡¡¡¡INTRODUCTION

¡¡¡¡M¨¹ller 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 M¨¹ller cells and RPE resulted in the formation of proliferative membrane. In the present study, M¨¹ller cells were coª²cultured with RPE both under normoxic and hypoxic conditions to observe the effect of RPE on the proliferation and migration of M¨¹ller cells.

¡¡¡¡MATERIALS AND METHODS

¡¡¡¡Cell Culture  Newborn pigmented rabbits (8ª²10 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.

¡¡¡¡M¨¹ller 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 Dulbecco¬ðs Modified Eagle Medium (DMEM), supplemented with 200mL/L fetal bovine serum (FBS), at 37¡æwith 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 Sª²100 antibody. The secondª²to fourthª²passage 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 phosphateª²buffered saline (PBS), and a solution of trypsin (2.5g/L) was applied to the cup for 30 minutes at 37¡æ. 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¡æ 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 M¨¹ller Cells  Two assays were performed to observe the proliferation of M¨¹ller cells. One detected the change of M¨¹ller cells at various time points; another detected the change of M¨¹ller cells under various conditions. Hypoxia was achieved using CoCl2 at a concentration of 200¦Ìmol/L.
Assay for various time points  Transwell system (0.4¦Ì£í) was used to develop a coª²culture system[3].  In the coª²culture group, the RPE (200¦ÌL, 5¡Á104/mL) were cultured in the upper wells (insert), while the M¨¹ller cells (500¦ÌL, 5¡Á104/mL) were cultured in the lower wells. As controls, there were no cells cultured in the lower wells[4].  In both the coª²culture group and control group, M¨¹ller 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 coª²culture group at various time points£¨ÂÔ£©

¡¡¡¡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 (0.4¦Ì£í) was used to develop a coª²culture system. In the coª²culture group, the RPE cells (200¦ÌL, 5¡Á104/mL) were cultured in the upper wells (insert), while the M¨¹ller cells (500¦ÌL, 5¡Á104/mL) were cultured in the lower wells. There were no cells cultured in the upper wells in the M¨¹ller only group. The proliferation of M¨¹ller 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 M¨¹ller Cells  Two assays were performed to observe the migration of M¨¹ller cells. One was conducted to detect the change of M¨¹ller cells at various time points, and the other was conducted to observe any change of M¨¹ller cells under various conditions. Hypoxia was achieved by use of CoCl2, with a concentration of 200¦Ìmol/L.

¡¡¡¡Assay for various time points  Transwell system (8¦Ì£í) was used to develop a coª²culture system. In the coª²culture group, the M¨¹ller cells (200¦ÌL, 5¡Á104/mL) were cultured in the upper wells (insert), while the RPE cells (500¦ÌL, 5¡Á104/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 phaseª²contrast microscope ¡Á400. 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 (8¦Ì£í) was used to develop a coª²culture system. In the coª²culture group, the M¨¹ller cells (200¦ÌL, 5¡Á104/mL) were cultured in the upper wells (insert), while the RPE cells (500¦ÌL, 5¡Á104/mL) were cultured in the lower wells. There were no cells cultured in the lower wells in the M¨¹ller 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 phaseª²contrast microscope ¡Á400. In addition, the average number of cells under three observed fields was calculated under a phaseª²contrast microscope ¡Á400. 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 M¨¹ller cell proliferation in factorial design study(ÂÔ)

¡¡¡¡Time was treated as the withinª²subject factor and the group as the betweenª²subject 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 Sª²100 immunoª²histochemistry. Under transmission electron microscope (TEM), cells presented enriched cytoplasm, large nuclei and 8ª²10nm characteristic midª²silks. 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 appeaª²rance and the expression of yellowª²brown through cytokeratin immunocytochemistry, the RPE cells were confirmed.

¡¡¡¡Proliferation of M¨¹ller Cells  Proliferation of M¨¹ller cells in the control group and the coª²culture 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 coª²culture group was found.

¡¡¡¡A 2¡Á2 factorial design study (condition [CO]: normoxia, hypoxia; culture [CU]: M¨¹ller cells and RPE, M¨¹ller cells only) showed that the main effects both of CO and CU were significant for proliferation of M¨¹ller cells, whereas the interaction term (CO¡ÁCU) was not significant. The MTT results under different culture conditions are shown by A values in Table 2 and Figure 1.

¡¡¡¡Migration of M¨¹ller Cells  The number of M¨¹ller 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 coª²culture group.Table 3  Number of M¨¹ller cells migrating in the control group and the coª²culture group atvarious time points(ÂÔ)Table 4  Migration of M¨¹ller cells in a factorial design(ÂÔ)

¡¡¡¡A 2¡Á2 factorial design study (culture [CU]: M¨¹ller cells and RPE, M¨¹ller cells alone; condition [CO]: normoxia, hypoxia) showed that the main effects both of CU and CO were significant for migration of M¨¹ller cells, whereas the interaction term (CU¡ÁCO) was not significant. Table 4 and Figure 2 showed the migration of M¨¹ller cells in the factorial design.

[1] [2] ÏÂÒ»Ò³