Effect Of Hyperglycemia On Late Endothelial Progenitor Cells From Human Peripheral Blood And The Effect Of Metabolic Memory

Background Endothelial Progenitor Cells(EPCs)are originated in the bone marrow and exist in the circulation of blood in a small amount of way.They can proliferate, differentiate to be endothelial cells in peripheral blood. Vascular endothelial dysfunction is the important initiating factor in the development of diabetic angiopathy. Hyperglycemia environment can lead to the ability decreased of adhesion, proliferation, migration, vascularization of endothelial progenitor cell, and can also lead to the function of endothelial cell disorder, cause a series of vascular complications. Previous studies showed that early strict control of glucose levels may delay the development of diabetes vascular lesions, but in the end it is poor earnings.Patients still have higher risk of vascular lesions. We aim to isolate EPCs from peripheral blood from healthy adult and identify late EPCs.Then simulate to build hyperglycemia metabolic memory. From the function of adhesion, proliferation, migration, angiopoiesis of late EPCs to observe whether there is a hyperglycemia metabolic memory phenomenon in late EPCs.Our study tend to provide theoretical and practical basis to intervene vascular lesions occur in clinical. And also provide new ideas for the treatment of diabetes vascular lesions in the future.Methods Our experiment collected peripheral blood from healthy adult volunteers,then adopted the method of density gradient centrifugation to separate and obtain mononuclear cells(MNC).Culture cells in vitro by special medium and observe its growth conditions under the microscope.On condition that cells appear tridimensional pebbles form proceed cells passage culture. At the same time, proceed immunochemical staining, identification the expression of CD31, CD34, KDR, CD133 of cells during this period. And detect the ability of those cells uptaking acetylated LDL(Ac-LDL) and combining with plant lectins(UEA-1).Then establish hyperglycemia memory model, divide cells into three groups namely: normal glucose group(5.5 mmol/L for treatment 6 days), high glucose group(30 mmol/L for treatment 6 days), hyperglycemia memory group(30 mmol/L for treatment 6 days then 5.5 mmol/L for treatment 6days). Adhesion test, bromine salt determined by MTT colorimetry,Transwell chamber, angiopoiesis experiments were used to measure the ability of adhesion, migration, proliferation, tube formation respectively.Then process statistical analysis.Results1. The new separation cells from human peripheral blood mononuclear are bright and rounded, suspended in the medium. When culture four days in vitro, many cells are adhering to the wall, volume be large,morphology become long fusiform. Cells culture to 14 days or so begin to present tridimensional pebbles morphology, proliferation ability get strong. Cells of this period can continue to passage cultivation, also can express CD31, CD34, KDR, CD133 and uptake acetylated, combine with plant lectins. Hint this class cells is proliferating late endothelial progenitor cells.2. In vitro, late EPCs under the hyperglycemia(glucose concentration as30 mmol/L) condition last a certain time(6 days), their ability of adhesion, proliferation, migration, tube formation are decreased comparing with continuous normal glucose group. The difference is statistically significant.3. Late EPCs in certain glucose concentration(30 mmol/L) under the cultivation of a certain time(6 days), then adjust medium to normal glucose concentration(5.5mmol/L), the function of late EPCs been impaired comparing with duration high glucose group(30 mmol/L).Suggest late EPCs may exist hyperglycemia metabolic memory phenomenon.Conclusion1. Late EPCs can be isolated from human peripheral blood and cultivate by using density gradient centrifugation2. Hyperglycemia can lead the ability of adhesion, proliferation, migration,tube formation decrease of late EPCs. This may be associated with the damage of hyperglycemia act on endothelial cells.3. Late EPCs from peripheral blood in the state of high glucose after a certain period of time, the function of cell are descended even if correct glucose concentration subsequently. Late EPCs may exist hyperglycemia metabolic memory phenomenon.