Obiective
lt;brgt;To investigate the change of the activity of proliferation in cultivated Muuml;ller cells treated by advanced glycation endoproducts (AGEs), and the effect of these changes on expression of occludin in bovine retinal vascular endothelial cells (BREC).
lt;brgt;Methods
lt;brgt;The cultivated Muuml;ller cells were devided into normal growth group and cultured with AGEs group. The cultured BREC were devided into 4 groups:group 1, without any medium; group 2, with normal growth Muuml;ller cell medium (MCM); group 3,MCM treated by AGEs; group 4, without cell as the control. Enzyme-linked immuno sorbent assay was used to detect the content of occludin in the medium in the 4 groups.
lt;brgt;Results
lt;brgt;The content of expression of occludin was the most in group 2, less in group 1, and the least in group 3.
lt;brgt;Conclusion
lt;brgt;AGEs may promote the abnormal proliferation of Muuml;ller cells and inhibit the expression of occludin in BREC.
lt;brgt;(Chin J Ocul Fundus Dis, 2006, 22: 28-30)
ObjectiveTo observe the effect of polypyramidine tract binding protein-associated splicing factor (PSF) towards advanced glycation end products (AGEs) induced the apoptosis of Müller cells in vitro.MethodsExperimental study. Müller cells were cultured and divided into groups according to the project design, plasmid enhanced green fluorescent protein-PSF were transfected into the cells to achieve the overexpression of PSF Müller cells in vitro, then cells were exposed to AGEs and the Morphological changes were observed by HE staining and Hoechst 33258 staining while the survival rate of cells were detected by MTT assay. The effects of PSF on AGEs-induced Müller apoptosis was measured by Cell Death Detection ELISA kit. Meanwhile, 2′,7′-dichlorofluorescin diacetate staining was performed to monitor the protective effects of PSF on AGEs-induced Müller cells ROS.ResultsThe morphology of cells in normal group was full and the cytoplasm staining was uniform. In N+AGEs group and Vec+AGEs group, cell volume decreased, cytoplasm was dense and concentrated, and eosinophilic staining was enhanced. The cell morphology of PSF+AGEs group was still full, with uniform cytoplasm staining and uniform nucleus staining. The viability of N+AGEs group, Vec+AGEs group and PSF+AGEs group were 0.42±0.11, 0.35±0.12 and 0.68±0.12. The apoptosis values were 1.08±0.16, 0.96±0.20 and 0.44±0.08. The intracellular ROS levels were 28 833.67±3 550.06, 28 356.67±4 854.81, 186 163.00±382.54. Compared with N+AGEs group and Vec+AGEs group, the cell viability of PSF+AGEs group was significantly improved (F=20.65, P=0.000), cell apoptosis value (F=43.43, P=0.000) and intracellular ROS level (F=18.86, P=0.000).ConclusionPSF overexpression play a protective role in AGEs-induced apoptosis by inhibiting the production of ROS in Müller cells.
Objective
To study the effects of advanced glycation end (AGEs) products induced by bovine serum albumin (BSA) on the survival and the morphology of bovine retinal endothelial cells (BREC) and pericytes (BRP).
Methods
BSA with the final concentration of 50 mg/ml was incubated in PBS, containing 500 mmol/L D-glucose, for 12 weeks under 37℃. AGEs-BSA was purified by Sephacryl S-300 column chromatography and was confirmed by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). The concentration of AGEs-BSA was determined by the method of commassie protein assay. In order to detect the toxic effects of AGEs-BSA on cultured BREC and BRP, groups of AGEs-BSA and BSA with different concentration and untreated control were set up. Phase contrast microscope was used to observe the effect of AGEs-BSA and BSA (with the concentration of 500mu;g/ml and actuation duration of 48 hours) on morphology of BREC and BRP.
Results
As the dosage of AGEs-BSA increased, the number of inhibited cells increased. When the concentration of AGEs-BSA was 500mu;g/ml, the inhibited BREC in AGEs-BSA group was (72.8plusmn;15.9)% of which in untreated control group, and the inhibited BRP was (64.8plusmn;9) % of which in untreated control group. AGEs-BSA with low concentration promoted the proliferation of endothelial cells, but there was no significant difference between AGEs-BSA and the control group (P=0.231). Inhibited proliferation and abnormal morphology were seen under the phase contrast microscope while the normal morphology of cells was found in BSA and control group.
Conclusion
AGEs-BSA with the high concentration may inhibit the growth of both BREC and BRP, which leads the loss of BRP and damage of vascular function. These results suggest that nonenzymatic glycosylation plays a major role in diabetic complications.
(Chin J Ocul Fundus Dis, 2006, 22: 11-15)
Objective
To investigate the effect of advanced glycation end products (AGEs) on the catalase activity and the levels of malondialdehyde in cultured bovine retinal capillary pericytes (BRPs), and to investigate the relationship between oxidative stress and diabetic retinopathy.
Methods
Cultured BRPs were exposed to AGEs (0, 8, 32, 125, 500, 2 000 μg/ml) for four days. Activity and the levels of catalase and malondialdehyde in cultured BRPs were examined by spectrophotometry.
Results
AGEs decreased the catalase activity, whereas increased the levels of malondialdehyde of cultured BRPs in a dose-dependent manner (r=-0.714, r=0.748, P＜0.01).There were significant differences between BRPs cultured in 32 μg/ml AGEs and in control group (P＜0.01), while no significant differences between BRPs cultured in non-glycated bovine serum albumin and absence of bovine serum albumin were found.
Conclusion
Oxidative stress may be one of the reasons why the pericyte disappears in diabetic retinopathy.
(Chin J Ocul Fundus Dis, 2002, 18: 143-145)
ObjectiveTo summarize the research progress of the effects of high glucose microenvironment on the biological activity of adipose-derived stem cells (ADSCs).MethodsThe literature on the high glucose microenvironment and ADSCs at home and abroad in recent years was reviewed, and the effects of high glucose microenvironment on the general characteristics, differentiation potential, angiogenesis, and nerve regeneration of ADSCs were summarized.ResultsThe accumulation of advanced glycosylation end products (AGEs) in the high glucose microenvironment led to changes in the biological activities of ADSCs through various pathways, including cell surface markers, proliferation, migration, multi-lineage differentiation, secretory function, and tissue repair ability. The ability of ADSCs to promote angiogenesis and nerve regeneration in high glucose microenvironment is still controversial.ConclusionHigh glucose microenvironment can affect the biological activity of ADSCs, and the effect and mechanism of ADSCs on angiogenesis and nerve regeneration in high glucose microenvironment need to be further studied.
Objective
To investigate the effects of glycation pro ducts on growth and cytosoic free calcium (［Ca2+］i) of bovine retinal capillary pericytes.
Methods
The changes of growth and ［Ca2+］i of bovine retinal pericytes,which were cultured in early glycation products of bovine serum albumin (EG-BSA) and advanced glycation end products of bovine serum albumin (AGE-BSA),were studied by counting cell numbers,MTT colorimetric assay,［3H］thymidine incorporating,and fluorescent indicator fura-2 acetoxymeth1 ester (Fura-2AM).
Results
The number of alive pericytes in groups of EG-BSA and AGE-BSA were 17.87plusmn;2.36 and 14.77plusmn;3.72 which comparing with their control groups (20.54plusmn;0.82 and 20.31plusmn;0.93)were de creased 13．00% and 27．00% (Plt;0.01) by counting cell numbers on a counting plate after four days.The results were 0.4619plusmn;0.0946 and 0.3884plusmn;0.1031 which comparing with their control groups (0.5236plusmn;0.0539 and 0.5227plusmn;0.0519)were decreased 12．00% and 25.70% (Plt;0.01) by MTT colorimetric assay.Amount of ［3H］thymidine incorporating in groups of EG-BSA and AGE-BSA were 39450.16plusmn;887 0.68 and 33667.85plusmn;10581.70 which comparing with their control groups (56373.63plusmn;2317.97 and 56542.04plusmn;1961.23)were decreased 30．00% and 40.40% (Plt;0.01).The ［Ca2+］i concentration of pericytes in groups of EG-BSA and AGE-BSA were (129.55plusmn;30.41) nmol/L and (179.71plusmn;56.69) nmo l/L which comparing with their control groups ［(79.70plusmn;6.94) nmol/L and (83.plusmn;6.39) nmo l/L］ were increased to 163．00% and 214．00%.
Conclusion
Both EG-BSA and AGE-BSA can inhibit the proliferation and DNA syntheses of retinal capillary pericytes,and increased ［Ca2+］i concentration in pericytes,especially the AGE-BSA.
(Chin J Ocul Fundus Dis，2000，16：139-212)
ObjectiveTo observe the protective effect of polypyrimidine bundle-binding protein-related splicing factor (PSF) over-expression on RPE cell injury induced by advanced glycation end products (AGEs).MethodsThe human RPE cells cultured in vitro were divided into three groups: normal control group (N group), blank control group (N + AGEs group), empty vector control group (Vec + AGEs group), and PSF high expression group (PSF + AGEs). group). RPE cells in N group were routinely cultured; RPE cells in N + AGEs group were only transfected but did not introduce any exogenous genes combined with AGEs induction; Vec +AGEs group and PSF + AGEs group were transfected with pcDNA The empty vector or pcDNA-PSF eukaryotic expression plasmid was introduced into RPE cells and induced by AGEs. Except the N group, the other 3 groups of cells were transfected accordingly, and were stimulated with 150 μg/ml AGEs for 72 h after 24 h. HE staining and Hoechst 33258 staining were used to observe the effect of high PSF expression on the morphological changes of RPE cells; ROS level detection was used to analyze the effect of PSF high expression on the ROS expression of RPE cells induced by AGEs; MTT colorimetric method was used to detect the high PSF expression Effects on the viability of RPE cells; Western blot was used to detect the effects of different time and dose of PSF on the expression of heme oxygenase 1 (HO-1).ResultsHE staining and Hoechst 33258 staining observation showed that the cells in group N were full in shape, the nucleus was round, the cytoplasm was rich, and the staining was uniform; the cells in N + AGEs group and Vec + AGEs group were reduced in size, the eosinophilic staining was enhanced, and the nucleus was densely densely stained. Pyrolysis and even fragmentation; the morphology of cells in the PSF + AGEs group was still full, the cytoplasm staining was more uniform, and the nucleus staining was uniform. The results of MTT colorimetry showed that high expression of PSF can effectively improve the viability of RPE cells, but this effect can be effectively antagonized by ZnPP, and the difference is statistically significant (F=33.26, P＜0.05). DCFH-DA test results showed that compared with the N + AGEs group and Vec + AGEs group, the ROS production in PSF + AGEs group decreased, the difference was statistically significant (F=11.94, P＜0.05). Western blot analysis showed that PSF protein up-regulated HO-1 expression in a time- and dose-dependent manner. The relative expression level of HO-1 at 24, 48, and 72 h after PSF protein was significantly higher than that at 0 h, and the difference was statistically significant (F=164.91, P＜0.05). The relative expression level of HO-1 under the action of 0.1, 0.5, 1.0, 1.5, and 2.0 μg PSF protein was significantly higher than 0.0 μg, and the difference was statistically significant (F=104.82, P＜0.05).ConclusionPSF may inhibit the production of ROS by up-regulating the expression of HO-1, thus protecting the RPE cells induced by AGEs.
