Effect Of Glycated Basic Fibroblast Growth-2 On The Biologic Activity Of Endothelial Progenitor Cells Originated From Rat's Bone Marrow In Vitro

Objective: According to related reports , it has been demonstrated clearly that neovasculorization and micro vascular function are impaired in diabetes animal models of diabetes mellitus and it has been shown that in the high sugar environment, the quantity and the function of endothelial progenitor cells (EPC) reduced which be regarded to maintaining the endothelial cell's integrity, promoting the neovasculorization. As a consequence of increased glucose availability, free amino groups of proteins undergo a nonenzymatic reaction with reducing sugars, leading to the formation of advanced glycation end-products (AGEs). This posttranslational modification of proteins is responsible for the thickening of the capillary basement membrane, a hallmark of diabetic microangiopathy. It also is one of the causes of the diabetic trunk and capillaries complication. Besides Macro-molecule protein will be glycated in the high sugar environment , basic fibroblast growth factor (FGF-2) which takes parts in mitogenic activity of cell and promote the angiogenesis of EPCs also change to be gFGF-2 with the process of FGF2 non-enzymatic glycation .It affects the function of EPC,further more, it also be responsible for the digression of angiogenesis . Therefore, the major aims of the present study were to evaluate the effect of hyperglycemia on fibroblast growth factor-2 (FGF2)-induced angiogenesis by observeting the number and biological ability of EPCs and to determine whether FGF2 non-enzymatic glycation occurs in hyperglycemia. Methods: 1.Make and identify the gFGF-2: recombinant FGF2 was dissolved in phosphatebuffered saline (PBS), pH 7.4, at 10 ug/ml final concentration and immediately frozen at–70°C. Then aliquots of FGF2 were incubated at 37°C for 48 hours with 250 mmol/L fructose. Analysis of glycated proteins by matrix-assisted laser desorption/ionisation mass spectrometry (MALDI) 2.Isolation,cultivate and identification of the EPC in bone marrow: Mixed bone marrow with equal amount PBS solution in the 15 ml volume centrifuge tube prepared lymphocyte isolation solution(1.083g/ml).After density gradient centrifugation with 2000r / min×25min ,isolated the middle white layer to another test tube, added 5 times volume of M199 and centrifuge with 1500 r/min×5min.Then to ablution twice,abandoned the supernatant and added M199 for suspending the cells. Cultivated cells in M199 solution cultivate plate prepared with collagen in 1×106/L density and incubation in 37℃,5%CO2 incubator. Replace the cell cultivate medium in every 3-4 days and abandon cells suspending in the medium. Observe the shape/growth/multiplication condition consequently by inverted phase contrast microscope. Separate the EPCs after 4 days , and go on cultivate with three groups for another 3 days , FGF-2(150ng/ml), gFGF-2(150ng/ml) and blank group(basic culture fluid without anything). Identify EPC with CD34,CD133,VEGFR-2,ac-LDL and UEA-1 by immunohistochemistry and immunofluorescence and count the number in different groups. 3.Biological ability test of EPCs in the bone marrow Collect attached cells after 4 days by trypsinization then cell were separate in three group , incubated at 37℃for 72 hours with gFGF-2,FGF-2and basic culture fluid without anything. Further more, Collected cells after cultivated 7 days to evaluate angiogenesis activity with vitro angiogenesis kit;, test the attachment ability; Determine migration ability of EPC with the modified Boyden cave method; Determine reproductive activity by MTT method ; Determine amount of EPO, GM-CSF and IL-8 in cell culture solution by radioactivity immunization. Results: FGF-2 is successfully glycated to gFGF-2 by the examination of protein fingerprint analysis. Angiogenic capability test of gFGF-2 group and FGF-2 group in vitro indicate that FGF-2 glycated reduces angiogenic activity (P<0.05); The number, adhesion ability, migration ability;reproductive activity;amount of EPO, GM-CSF GM-IL-8 of EPC of gFGF-2 group is lower than FGF-2(P < 0.05). Conclusion: The number and the biologic activity of Endothelial Progenitor Cells, which originated from Rat's bone barrow in vitor, descrese when cultivated in gFGF-2 and the angiongenesis capability in vitro also obviously reduced.Thus, gFGF-2 may be a reason which lead to the impairment of angiogenesis in diabetes mellitus.