Effect Of Xue Fu Zhu Yu Decoction On Structure、Mobilization And Function Of Rat Endothelial Progenitor Cells From Bone Marrow

[ Background and Objective]Vascular endothelial progenitor cells(EPCs)are the precursors of endothelial cells(EC). EPCs can be mobilized to peripheral blood, and then migrate, home to the sides of new blood vessel formation, in which they can proliferate, differeatiate to EC and form new blood vessels eventually. EPCs play an important role in ischemia disease. The results of the research about EPCs gives us a new alternative choice for therapy of ischemia disease.According to the thery of traditional Chinese medicine(TCM) Xue Fu Zhu Yu Decoction (XFZYD) which is from <<Errors in Medicine Corrected>> of great physician Wang Qing ren possess the capability to promote blood flow, not to waste blood, removing blood stasis and forming new. Clinically, XFZYD can improve microcirculation, treat coronary heart coronary heart disease, thrombus phlebophlogosis and retina phlebitis thrombus, and so on. We found XFZYD can significally promote angiogeneesis in screening experiment of chick embryo allantocherion.So we investigated the effects of XFZYD on the structure, mobilization and the ability of proliferation,migration, uptaking Dil-acLDL, adhesion and capillary tube formation in vitro of EPCs from bone marrow. And identify the effects of promoting angiogenesis.[Methods]1 Culture and identification of rat EPCs from bone marrowBone marrow mononuclear cells(BMMNCs) were harvested from bone marrow by flushing rat femur and tibia and were isolated by density gradient centrifugation, plated on culture dishs coated with gelatine,maintained in DMEM culture solution. After 4d of culture, nonadherent cells were removed by washing with DMEM, new medium was applied and the culture was maintained.EPCs were characterized as adherent cells positive for Dil-acLDL uptaking by fluorescent inverted microscope, and demonstrated by expressing CD31, CD34, vWF using flow cytometry and immunity tissue chemistry, futher observe the structure of EPCs by electron microscope. 2 Effects of XFZYD on the mobilization、structure and function of EPCsThe concentration was studied by estimating vascular endothelial(VEGF) secretion using euzyme-linked immunosorbent assay(ELISA), nitric oxide (NO) concentration using nitrate reductase. After cultured for 11d, EPCs were randomized into 12 groups, every group containing serum at final concentrations of 5%, 10% and 15%. After different periods of culturing in vitro 24h, 48h and 72h, observe the structure by electron microscope, the ability of cell proliferation,migration and adhesion was assessed by MTT assay, fluorescent inverted microscope, transwell chamber and cell counting respectively. The phenomenon of uptaking Dil-acLDL and capillary tube formation in vitro was observed on the microscope.[Results]1 Culture and identification of rat EPCsThe lately-separated bone marrow mononuclear cells(BMMNCs) are round.Parts of the cells start attaching after 48 hours. 3 days later, the cells were able to form cell’colonies, arranged in lines at 5 days, and almost cells were shuttled from 7 days. At 11 days,it can form blood vessel structure. The attached cells were positively stained for CD31、VEGFR-2 and vWF , had taken Dil-acLDL, observed rough surfaced endoplasmic reticulum, mitochondrion, golgi body by electron microscope.2 Effects of XFZYD on the mobilization of EPCsXFZYD increase the concentration of NO in a dose-dependently manner. Compared to blank control serum group, the concentration of VEGF was set up in high dose of medcine group, G-CSF was cut down, it can increase GM-CSF and SDF-1 in middle dose, and the concentration of VEGF were cut down in middle and low doses.3 Effects of XFZYD on the structure、function of EPCsThe function of proliferation,migration, differentiation,adhesion, uptaking Dil-acLDL and capillary tube formation in vitro of endothelial progenitor cells were remarkably increased with XFZYD, which was most prominent at the concentration of high and middle doses.[Conclusion]1 Through the method of ficoll density gradient centrifugation can be isolated and culure bone marrow-derived EPCs.2 XFZYD increase the concentration of VEGF, GM-CSF,SDF-1 and NO.3 XFZYD aslo increase the ability of proliferation,migration, differentiation, adhesion, uptaking Dil-acLDL and capillary tube formation in vitro of endothelial progenitor cells.4 XFZYD could induces EPCs taking part in angiogeneesis. It’s likely to be a newmechanism of EPCs for therapy ischemic disease.