| Objective 1. To investigate nonenzymatic glycosylation process of Human serum albumin (HSA) incubated with high concentration of glucose and the inhibition effect of several flavonoids on the protein nonenzymatic glycosylation in vitro. 2. To investigate effects of AGEs on the proliferation and apoptosiss in bovine retinal microvascular pericytes and human mesangial cells as well as the regulation of extracellular matrix components synthesis of human mesangial cells. 3. To determine the pathogenic role of AGEs in vivo and to develop a new animal model system for the study of diabetic microangiopathy. Methods 1. HSA was incubated with D-(6-3H)-glucose at 3TC for eight days and the protein nonenzymatic glycosylation was determined to study the process of the reaction by measuring the incorporation of D-(6-3H)-glucose into protein. 2. Glycated proteins in vitro were achieved by bovine serum albumin incubating with 200mM glucose for 60 days. After incubation of BRP or HMC with 0.000I,0.00l,0.01,0.I,lmmolll of AGE-BSA from 24 to 96 h, we detected the degree of cell proliferation by MTT assay , and the degree of cell apoptosis by staining with Hoechst 33258 or flow cytometric assay. Levels of extracellular matrix components of HMC including collagen IV and Laminin were measued by radioimmunoassay. 3. AGE-modified bovine serum albumin was administrated to healthy nondiabetic rats or in combination with AGE-crosslinking inhibitor aniinoguanidine for 40 days of AGE treatment. Serum malondialdehyde (MDA) levels and antioxidative enzyme activity, including superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), were determined by spectrophotometry. Serum AGEs levels were assayed by specific ELISA. After perfusion with saline and buffered formaldehyde, The retinas isolated from animals were first fixed and then digested with typsin. The preparations of typsin-digested retinal blood vessels were stained with periodic acid-Schiff technique (PAS), and counter-stained with hematoxylin to evaluate changes of retinal capillaries. Immunohistochemical AGE and VEGF localization was explored on the complete retinal vascular tree after isolation by typsin digestion using a rabbit antiserum against AGE and a Strept Avidin-Biotin Complex system. Results 1. After 6 days of incubation at high glucose concentration, incorporation of D-(6-31I)-glucose into protein was significantly increased. These changes can be significantly inhibited by the additions of aminoguanidine (0.5mg/mi), kakonein(4.OmgIml) gingko flavone(4.OmgIml), crataegin group does not show the inhibition effect. 2. AGEs time- and concentration-dependently inhibits the proliferation of BRP. There were no significant differences between BRP cultured for 24 and 48 h with 0.0001,0.001 mind/I AGEs and in controls. However, proliferation was significantly inhibited by 0.01,0.1 ,lmmol/l of AGEs (compared with BSA group,p<0.05). The IC50 is 0.Ol5mmol/1 after 96h of exposure to AGEs. Apoptosis of BRP was induced by 24 h of exposure to AGEs. The proportions of apoptotic cells cultured with 0.001,0.01,0.1 mmol/l of AGEs were 5.08%,8.44%,18.61% respectively. Compared with BRP, the inhibitory effects of AGEs on the proliferation of HMC were relatively mitigated. After 24 an... |
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