Treatment Of Early Rabbit Femoral Head Necrosis With Epigenetic Modified EPCs

Background In the present, the cultured endothelial progenitor cells (EPCs) was generally obtained from promoting primary mononuclear cells differentiation, but the cell amount was limited and not enough to support large scale experiment. In view of the difficulty of EPC culture, until now there is no commercial EPCs cell lines established improving efficacy of EPCs differentiation and proliferation is the key issue of EPCs based on treatment of femoral head necrosis.Objective Establish a simple method to culture EPCs and meet the further study of EPCs biological functions and fulfill the needs of EPCs based on cell therapy, so that EPCs based on regenerative science is no longer constrained in the methodology.Methods Separate mononuclear cells from human umbilical cord blood by Ficoll density gradient centrifugation, and use the modified Hill colony formation to culture EPCs. Observe EPCs morphological changes by microscopic, detect EPCs uptake of Dil-Ac-LDL-and FITC-UEA1 by immunofluorescence, use flow cytometry to evaluate the expression of EPCs surface markers:CD133/KDRand the CD34/KDR, measure EPCs proliferative capacity by MTT assay.Results Using the modified Hill colony formation to culture EPCs, typical of EPCs colony formation were observed under a microscope. The results of immunofluorescence showed that 99.7%cells is double positive, which confirmed that EPCs were success-fully cultured. The result of Flow Cytometer showed that EPCs surface markers CD 133/ KDR and the CD 34/KDR expression rates were 52.30%±7.43% and 54.77%±3.02%. The MTT results showed that the cells showed a trend of steady growth after inmono-nuclear cells differentiate into EPCs.Conclusion EPCs surface antigen expressions were more typical, which confirmed that early EPCs was formed. Modified Hill colony formation of cultured endothelial progenitor cells, can be a sufficient methods to culture EPCs by increasing EPCs proliferation and differentiation capacity, which can provide a strong support for the further study EPCs biological behavior. Background The etiology of avacular necrosis of femoral head (ANFH) is complex. But now the pathogenesis is not exactly known. It is considered that ANFH is caused by many environmental factors in or ex vivo. And there is no therapy to reverse the pathological development of ANFH. The decrease of the number and function of endothelial progenitor cells (EPCs) was observed in our initial experiment and clinical observation. The CXCR4 play an important role in EPC's migration and angiogenesis. And the epigenetic research developed quickly in recent years has proved the gene's epigenetic modification is the bridge which links the environment and gene. So the research to the epigenetic modification of CXCR4 gene in EPC is important to finding a new therapy to reverse the pathological development of ANFH.Objective To up-regulate the CXCR4 gene expression in EPC via DNMT inhibitors and HDAC inhibitors. To detect EPCs number and function after CXCR4 expression was upregulated. In order to improve the efficacy of the the EPCs treatment of ischemic disease, and find a fundamental way to reverse of ANFHMethods After being isolated by Ficoll density-gradient centrifugation from human umbilical cord blood, MNCs were cultured in growth medium supplemented VEGF (10 ng/ml) and FGF-basic (10 ng/ml) for a week to obtain EPCs. After being characterized, EPCs obtained after 6 days' cultivation were treated with TSA and DAC combinedly or respectively at different concentrations for 48h kept in the dark in incubator. Then the MTT assay was performed to detect the cell number of EPCs and relative viability. Western blot and Real-time quantitative PCR was performed to analyze the expression of CXCR4. And methylation-specific PCR (MSP) was performed to analyze the CpG island methylation status in the CXCR4 gene. The transwell assays were carried out to detect the migration capability of EPCs.Results The results of MTT reveal the absolute number of EPCs was increased. As the drug concentration and intervence time increased, the cell number increased more slowly. The cell's relative viability reduced as the drug concentration and interfere time increased. The results of the Western blot and Real-time quantitative PCR analysis show that the CXCR4 gene expression in EPC was up-regulated after treated with the drugs. The results of MSP reveal that the CpG islands are methylated in the CXCR4 gene in the control group. While treated with DAC or TSA, the CpG island methylation status in the CXCR4 gene was changed into unmethylation.Conclusion All the experiment results reveal that the CXCR4 gene expression in EPC can be up-regulated via epigenetic mechanism to enhance EPC's function in tissue reparation. Changing epigenetic modification of CXCR4 gene in EPCs could be a novel therapy to reverse the pathological development of ANFH. Background The avascular necrosis of femoral head (ANFH) is a progressive disease, the disease is difficult to reverse, the majority of outcome in patients with total hip replacement surgery. The studies found that EPCs can osteogenic differentiation, and enhance the ability of self-healing and the formation of blood vessels in ischemic tissue through paracrine potential for treatment of ANFH. A growing number of studies have shown that and ANFH have similar environmental factors of cardiovascular disease, epigenetic modification in the artery stenosis and atherosclerotic disease process plays a vital role. The above findings suggest that you can try to treat of ANFH through epigenetic modification of EPCs.Objective In this study, the epigenetic modification approach to improve the EPCs cell surface CXCR4 expression, and its application in ANFH animal models to test its therapeutic effect.Methods 50 New Zealand white rabbits adaptive feeding for 4 weeks after osteonecrosis of modeling, adaptive feeding during autologous EPCs enrichment, cryopreservation spare. Osteonecrosis of the model was evaluated by MRI testing five weeks later, modeling the success of New Zealand white rabbits were divided into three groups: control group, EPCs treatment group, epigenetic-EPCs treatment group. Respectively, saline, EPCs suspension, the suspension of the epigenetic modificated of EPCs was injected via the ear vein infusion.4 weeks after the detection of micro CT and histological sections, to evaluate the therapeutic effect.Results 36 in the 50 New Zealand white rabbits were evaluated to be AHFN by MRI, suggesting that early femoral head necrosis. The osteonecrosis rate is 72%. Micro-CT examination:CG:femoral head subchondral trabecular bone is destroyed, reducing the number of thinning; the EEG:a small number of femoral head cartilage trabecular bone is destroyed, the destruction of cartilage, trabecular bone volume is less than the control group but more than EG. Light microscope studies:CG:empty lacunae were more and trabecular became\thinning; EEG:the number of empty lacunae is small and trabecular thinning was not obviously; the EG is between CG and EEG.Conclusion The infusion of self-enrichment of body EPCs treatment strategies improved the ability of regional self-repair of femoral head necrosis and angiogenesis, and promoted tissue regeneration and repair. This may change the direction of early osteonecrosis of the pathological progress, and avoid osteonecrosis progressed to collapse of phase. Thus to make ANFH patients as possible to delay or do not need to do a total hip replacement. And there is a more option for the treatment of early ANFH.