| BACKGROUNDDiabetic retinopathy(DR) was one of the most serious complications of diabetes. There were many factors taking part in the development of DR, but it was not very clear how DR occurred. It was well known that high expression of iNOS and high level NO was one of reasons for pathological changes in DR. Müller cells were the important gliocyte in the retina, affecting the development of DR. P2 receptor, one of the subtypes of purinergic receptor, generally resided in various types of cells, with many important physiological functions. P2Y2 receptor was a subtype of P2 receptor, and there might be changes in expression of P2Y2 receptor during pathological conditions, affecting the development of many diseases.AIM1.To isolate, purify and culture rat Müller cells.2.To establish a high glucose model of Müller cells in vitro, and to observe the effects of high glucose on P2Y2 receptor.3.To observe the effect of suramin, one of nonselective inhibitors of P2 receptor, on iNOS of Müller cells under high glucose in vitro.METHODS1. Müller cell culture and identification:Eyeballs were taken from newborn rat within 1 week. Retinas were isolated and minced, digested in the trypsin solution, the suspension was filtered through a 53μm nylon grit. After the filtrate was centrifuged, mixed cells were resuspended in 20% FBS medium and seeded into culture flasks. The cells were purified by a modified digestion method for 2-3 times. The Müller cells of passage 3-4 were characterized for the expressions and localizations of GFAP and GS.2. Effects of high glucose on P2Y2 receptor of Müller cells:After exposed to medium without FBS for 24 h, The cells of passage 3-4 were exposed to various concentrations of 5.5 (normal control), 10, 25 and 50mmol/L glucose (high glucose) for 24 h and 72 h. The immunofluorescence staining and its intensity were then applied to measure the changes of P2Y2 receptor of rat Müller cells.3. Effects of suramin on iNOS of Müller cells under high glucose:After exposed to 5% FBS medium for 24 h, The cells of passage 3-4 were exposed to various concentrations of 5.5+0(normal control), 25+0, 25+0.05 and 25+0.1 groups(high glucose+suramin) for 24 h and 72 h. The immunohistochemistry staining and its density were then applied to measure the changes of iNOS of rat Müller cells.RESULTS1. The rat Müller cells were purified by digestion of the trypsin solution. The cells showed positive immunohistochemistry staining for GFAP and positive immunofluorescence staining for GS. The purity of passage 3-4 was greater than 90%, and its viability was greater than 90%.2. The fluorescence intensity for P2Y2 receptor was increased significantly in the cells exposed to high concentrations of glucose (10, 25 and 50mmol/L) for 24 h with the values of 31.05±7.06, 59.17±11.42 and 84.11±12.72, respectively, compared with that (26.60±5.15) of controls (P<0.01). However, at 72 h, the intensity was only increased significantly in the cells exposed to high concentrations of 25, 50mmol/L with the values of 75.34±11.26 and 96.43±15.55, respectively, compared with that (26.91±4.88) of controls (P<0.01). There was no difference between the intensity of 10mmol/L (26.24±5.40) and that of controls (P=0.574). There was significant difference between the data of 24 h and that of 72 h in total (F=92.439, P<0.01).3. Compared with the characters of the cells of controls (5.5mmol/L), that of high glucose (25mmol/L) and high glucose+suramin group (25mmol/L+0.05g/L, 25mmol/L+0.1g/L ) didn't change evidently, but gap of cells and floating cells of high glucose+suramin group increased, compared with that of controls. There were no significant immunohistochemistry staining for iNOS in intracytoplasm of controls , 25+0.05(24 h)and 25+0.1(24 h). Other groups showed positive immunohistochemistry staining for iNOS. The density of immunohistochemistry staining was increased significantly in the cells exposed to high concentrations of glucose (25mmol/L) for 24 h with the value of 110.03±71.92, compared with that (90.70±44.14) of controls (P<0.01). However, There were no differences between the density of 25+0.05 (91.58±41.22) and 25+0.1 (104.94±45.53) and that of controls (P=0.884 and P=0.018) for 24 h. The density of immunohistochemistry staining were increased significantly in the cells exposed to high concentrations of glucose (25mmol/L), 25+0.05 and 25+0.1 for 72 h with the value of 283.83±143.48, 133.69±99.51 and 113.88±55.41, respectively, compared with that (63.79±28.10) of controls (P<0.01). There was significant difference between the data of 24 h and that of 72 h in total (F=103.709, P<0.01).CONCLUSION1. High glucose could upregulate the expression of P2Y2 receptor of Müller cells in vitro.2. Suramin, one of nonselective inhibitors of P2 receptor, could downergulate the expression of iNOS of Müller cells under high glucose in vitro. Some subtypes of P2 receptor might promote the development of DR.The innovation of this experiment:The study observed firstly the change of expression of P2Y2 receptor of Müller cells under high glucose in vitro and the effect of suramin, one of nonselective inhibitors of P2 receptor, on iNOS of Müller cells under high glucose in vitro. Similar reseach has not yet seen in the literature. |
PDF Full Text Request