Study Of The Biological Characteristics Of Human Sources Of Precursor Cells Into The Body Skin

Part OnePhysiological characteristic of adult endothelial progenitor cells fromperipheral blood in vitro cultureBackground: The discovery of endothelial progenitor cells(EPCs)completely changed the notioon for angiogenesis process inadults. Meanwhile, researches for application of EPCs in the fields ofangiogenesis and tissue engineering were evoked and promising resultswere achieved. However, some confusing results about EPCs reportedrecently by some researchers make further investigation about biologicalcharacteristic of this kind of cells becomes essential.Objective: To examine the biological characteristic of adultEPCs, especially especially explore the phenotype, proliferative ability andfunctional anylysis possessed by this kind of cells.Materials and Methods: Mononuclear cells (MNCs) isolated fromperipheral blood by density gradient centrifugation were plated on humanfibronectin-coated culture plates and cultured in EGM-2 medium. Thesurface marker profile, proliferative capacity and functional properities ofEPCs were examined.Results: The growth curve showed the adherent spindle shape cells havelittle proliferative capacity. Although the cells could express somepremature endothelial lineage markers they could not no express mature EC markers and maintained to express of monocyte markers. Infunctional test, for the first time, we identified the monocytoid function ofEPCs by combining Dil-Ac-LDL and Indian ink uptake test. All the cellsare positive for the both tests throughout the culture. Another typicalcharacteristic of ECs so-called W-P body was not detected in EPCs. After4 days of culture,the cells showd no ability to form tube-like structures inangiogenesis assay in vitro.Conclusion: Collectivelly, although adult EPCs from peripheral bloodhave some EC lineage properties, they maintain typical monocytefuncition and have little proliferative capacity, More persuasive evidencemay be essential to prove the existence of "ture EPCs"with endothelialfunction,but without monocyte function. Part twoFeasibility of getting "ture EPCs"from bone marrow cells in vitrocultureBackground: Recent studies have reported the existence of "ture EPCs"inadults body.However, probably due to the rarity of true EPCs in peripheralblood, this findings are difficult to be repeated. It's well known that EPCsare derived from bone marrow,which contain more EPCs than peripheralblood,so obtaining the cells from bone marrow would offer morepersuasive evidence for the existence of the cells in adults.Objective: To examine the biological characteristics of human bonemarrow derived cells under endothelial growth promoting circumstanceand discuss the possibility to obtain "true endothelial progenitor cells"with strong proliferative capacity by culture of bone marrow cells invitro.Material and Methods: Human bone marrow cells were plated onfibronectin-coated dish and cultured in EGM-2 medium. The adherentcells to be obtained by this way underwent Dil-Ac-LDL uptake test andIndian ink uptake test. The cells cultured for thirty days underwent flowcytometry for analysis of surface markers and RT-PCR assay forexpression of eNOS.Results: The results showed that the primary cells contained someDil-Ac-LDL uptake positive cells,but these cells didn't show significant proliferative capacity and gradually losed following long term culture.After thirty days of culture, most of the cells were positive for themarkers of mesenchymal stem cells but showed neither the expression ofEC cell markers nor eNOS mRNA.Conclusion: We failed to obtain EPCs with significant proliferation byin vitro culture of bone marrow cells.What we obtain by this process aremesenchymal stem cells without tendeny to differentiate into lineage cells.endothelial lineage cells. How to acquire "true EPCs" from bone marrowin adults needs to be further investigated.