| Background and ObjectivesEndothelial progenitor cells (EPCs) are contained in cord blood, fetal liver, bone marrow, peripheral blood and other tissues. EPCs can promote angiogenesis in ischemic tissue, repair vessel injury and mitigate the over-hyperplasia of intima. In addition, they rarely lead to immune rejection response when applied in the treatment of EPCs transplantation. All these advantages of EPCs attract researchers'great concern. We can see in clinic that the decreased EPCs and its dysfunction exist in part of aging people and people with coronary heart disease, diabetes, hypertension and severe pulmonary hypertension. But we find a better effect of treatment when EPCs are transfused to the ischemic area. Peripheral blood is not only convenient to acquire, but also has rich sources, which makes the EPCs'culture from peripheral blood has wide perspective. However the EPCs cultured in vitro have many disadvantages, such as low self-proliferation, premature replicate senescence and so forth, which restrict the application of EPCs. It is of great importance to improve the culture methods, promote the EPCs'ability to adhere and migrate. Research demonstrates that atorvastatin can not only decrease the lipid level, restrict the proliferation and migration of vascular smooth muscle cells and reduce the C-reactive protein level, but also improve endothelial function. Therefore, we apply the atorvastatin to culture the EPCs isolated from human peripheral blood so as to observe its effects on the number and functions of EPCs cultured in vitro and at last explore a new road to improve the culture of EPCs in vitro.Materials and methods1. Isolation:The mononuclear cells (MNCs) were isolated from human peripheral blood by Ficoll density gradient centrifugation;2. Group:The cells were intervened with atorvastatin in a series of final concentrations of 0, 1×10-7mol/L,10-6mol/L,10-5mol/Land 10-4mol/L(A, B, C, D and E group);3. Culture and amplification:the mononuclear cells were symmetriced (cell density transferred to 5×106/L) and were plated on pre-fibronectin-coated culture dishes. After 7 days'culture, attached cells were collected;4. Identification:EPCs were characterized as adherent cells double positive for Dil-acLDL-uptake and lectin binding FITC-UEA-I by direct fluorescent staining under a laser scanning confocal microscope;5. The number of migrated cells in each group (A, B, C, D and E group) were counted under the inverted microscope by small chambers of modified Boyden chamber. While the number of adhesive cells in each group (A, B, C, D and E group) were counted under the inverted microscope by cultivation method of reattached cells. We made a comparison between the migrated and adhesive ability in each group.Results1. Total mononuclear cells (MNCs) were isolated from human peripheral blood by Ficoll density gradient centrifugation and purified by differential adherence.2. After 7 days'culture, cells demonstrating double-Positive for both FITC-UEA-1 and DiL-acLDL were identified as differentiating EPCs.3. Compared with the group A, the number of EPCs, adhesive and migrating EPCs of Atorvastatin treatment groups (B, C, D and E group) was increased significantly In Atorvastatin treatment groups, the number of EPCs, adhesive and migrating EPCs was on the rise with the increase of Atorvastatin's concentration Conclusions1. The method of Ficoll density gradient centrifugation is easy and simple. After the second adherence, the EPCs are purer than before. In addition, the EPCs' micro-environment does not change completely, which makes the EPCs better.2. Atorvastatin's intervening culture can be regarded as a kind of improved method on the culture of EPCs from human peripheral blood.3. Atorvastatin can increase the number of EPCs, and also improve adhesive and migrated functions of EPCs. These effects obviously depend on Atorvastatin's concentration. |
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