Identification Of Peripheral Blood Fibrocytes Cultured In Vitro And Its Chemotactic Funetions Stimulated By Human Dermal Microvascular Endothelial Cells

The peripheral blood fiberocytes(PBFCs) is a multi-differentiationpotential of cells found in recent years. It has the characteristics of fibroblast, bythe latest research proven to differentiate into myofibroblasts and vascularpericytes. Fiber cells in peripheral blood after tissue injury in the inflammation ofthe rapid migration to local wound, and then play it antigen-presentingcontraction wound role, a variety of cytokines and extracellular matrix synthesisand release as well as promoting wound neovascularization. We can consider thatPBFCs directed migration is an important foundation to play a role in woundrepair. Bone marrow-derived PBFCs by what factor or cell chemotaxis anddirected migration to the wound site. On this issue we still have no reports. Inaddition, the isolated peripheral blood fibroblasts in vitro culture,immunomagnetic beads currently used methods require screening of purifiedPBFCs. This method is expensive while the cell yield is lower, and is not conducive to the launching of the clinical application.Objectives:To explore a more user-friendly the peripheral blood fibroblasts cultured invitro method, understanding the in vitro PBFCs biological characteristics andfunctions of PBFCs cultured in vitro. To investigate whether human dermalmicrovascular endothelial cells(HDMECs) chemotaxis on PBFCs.Methods:Total peripheral blood leukocytes were isolated from human peripheralblood by centrifugation over Ficoll-Paque and cultured in DMEM supplementedwith20%fetal calf serum. Adhered cells were detected by immunocytochemis-try, flow cytometry and electron microscopy.Build up chemotactic model withtranswell to study the effection between PBFCs and HDMECs.Results:1. Total peripheral blood leukocytes were isolated from human peripheralblood by centrifugation over Ficoll-Paque and it contained huge amount oflymphocytes, monocytes and small amount of granulocytes.With the suitableculture condition, those cells could differentiated to PBFCs in about two weeks.2. Many factors can affect the culture of PBFCs,we considered thatdifferent drawing materials of blood,the density of leukocyte seeded,mediumchange time and the appropriate serum concentration are all important factors todetermine the success of culture. We selected the healthy vlunteers between20to45year-old, cultured in DMEM supplemented with pH value7.2~7.3,20%FBSmedium, seeded cells at5×106/cm2in a six pore plate. Four days after the firstexchange of medium after every three days the medium was changed once isPBFCs culture suitable conditions, the cells get PBFCs by cultivation of theabove conditions purity of up to80%more than able to meet the general requirements of the scientific research and experimental.3. Immunocytochemical staining of PBFCs showed that after10days culture,CD34+and COLⅠ+were strongly expressed, continuously cultured to28days,most CD34+lost, oppositely, COLⅠ+were still strongly expressed. The resultshowed that PBFCs differentiated into fibroblasts sustainably.4. The Transwell penetrating experiments confirmed that human dermalmicrovascular endothelial cells of peripheral blood fibroblasts chemotaxis, andlaid the foundation for further studies of the angiogenesis process PBFCs thedirectional migration and its mechanism.Conclusions:With the appropriate culture conditions exist in the adult peripheral bloodprecursor cells isolated in vitro, cultured fibroblasts differentiate into peripheralblood, and to maintain its biological characteristics to establish a more reliable,stable isolated and cultured by density gradient centrifugation. Initial grasp of thebiological characteristics of the in vitro culture process PBFCs table changes, andon this basis, the study confirmed that human dermal microvascular endothelialcells in peripheral blood fibroblasts chemotaxis.