| Bone marrow and peripheral blood of adults contain a special sub-type of progenitor cells which are able to differentiate into mature endothelial cells, thus contributing to neovasculartzation.These angiogenic cells have properties of embryonal angioblasts and were termed endothelial progenitor cells (EPCs). In general, three surface markers (CD133,CD34, VEGFR-2) characterize the early functional angioblasts, located predominantly in the bong marrow. Later, when migrating to systemic circulation EPCs gradually lose their progenitor properties and start to express endothelial cell markers like VE-cadherin, endothelial nitric oxide synthase and von Willebrand factor. The number of circulating EPCs in healthy adults is rather low and a varety of conditions or factors may futher influence the number.In the context of possible therapeutic application of EPCs recent basic and clinical studies employing these cells for neovascularization of ischemic organs have been published. Accordingly, we designed that (1) transplantation of autologous BM-EPCs and EPCs mobilization from bone marrow into systemic circulation may augment neovascularization in response to tissue ischemia in a rabbit model of unilateral hindlimb ischemia. (2) transplantation autologous peripheral blood endothelial progenitor cells for therapeutic angiogenesis in patients with lower limb Atherosclerosis obliterans.(3) characterization of EPCs culturing in vitro.In this study, understanding the angiogenic role of EPCs and characterization of EPCs culturing in vitro was our goal. Objective: To investigate the angiogenic role of mobilizing and transplanting endothelial progenitor cells in hindlimb ischemia model. Methods: Left hindlimb ischemic model was surgically induced in rabbit by cutting femoral artery and ligating its branches. 30 rabbits were devided into 3 group averagely—cntrolgroup,G-CSF group and EPC group. Bone marrow EPCs were transplanted into ischemic region in EPC group. EPCs were mobilized from bone marrow into peripheral blood by using rhG-CSF 10μg/kg/d×10d in G-CSF group. PBS buffer was injected into ischemic region in control group. During the experiment period, blood pressure ratio(L/R) ,blood flow volume of inner iliac artery by color Doppler flow imaging ,engery Doppler,microvascular density ,DSA and VEGF expression in skeletal muscle were adopted to evaluate angiogenesis. All data are presented as mean ±SD. Statistical significance was evaluated by means of ANOVA followed by q test for comparison between any 2 groups with SAS software. Statistical significance was assumed at a value of P<0.05. Results: After ischemicmodel developed, one rabbit died in each control and G-CSF group, the others survived to be executed. In first day, rabbits had claudication, pallor and low temperature in left hindlimb. In control group,there was necrosis in toes of two rabbits in 7th day, dry necrosis below the knee of left hindlimb occurred in one rabbit in 14th day, the others had slight claudication or remarkable muscle atrophy. In EPCand G-CSF group, they had normal function, but slight muscle atrophy of hindlimb. Detected blood flow volume of internal iliac atery by color Doppler , in control group,G-CSF group and EPC group, it rised from 9.210± 2.375 ml/min, 9.632±1.794 ml/min, 9.585±1.670 ml/min in 0 day to 11.999±1.873 ml/min,15.081±1.179 ml/min,17.901±1.483 ml/ min in 28th day respectively, compared with each other ,there were remarkable difference(P<0.05). Doppler blood flow signal enhanced by ultrasonic microbubbles contrast, EPC group had higher flow signal than G-CSF group. Immunohistochemical staining indicated more microvascular and VEGF protein expression in EPC group than G-CSF group, compared with control group, suggested remarkable difference. EPC group had more angiographilly detectable collateral vessels than G-CSF group.Blood pressure ratio was similar to the others. Conclusion: EPC transplantation and G-CSF mobilization EPC had remarkable angiogenic role, but there had more collateral vessel formation... |
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