Differentiation Of Rabbit BM-MNC Into Endothelial Cells In Vitro

Objectives The renewal of surgical therapy and pharmacotherapy has greatly improved the patient's condition who suffer from peripheral vascular diseases nowadays. But the extensive stricture of artery and severe limb ischemic diseases still perplex the vascular surgery scientific circle all the time. With the development of molecular biology and molecular genetics, utilizing transgenic technology to stimulate healthy and effective neovascularization in ischemic tissue , which is called therapeutic angiogenesis, becomes the hot spot and gives hopes to those patients. However, few clinical applications of gene transfer for therapeutic angiogenesis have been carried out because of a lot of difficulties, and one of them is the absence of a fine cell carrier with high-performance. Recently, circulating EPC has been discovered in adult peripheral blood and which took the assumption to be possible.In this topic,we aims to investigate the method of isolation and culture of rabbit EPC-enriched fraction of BM-MNC in vitro and obtain a steadily passaged cell population which differentiates into EC,providing foundation for the further step on gene therapy for severe hindlimb ischemic diseases with BM-MNC as cell carrier.Methods After aspirating BM from rabbit, endothelial progenitor cells(EPC)-enriched BM-MNC were isolated by centrifugation through a density gradient and cell depletion with dynabeads, and then cultured in Endothelial Growth Medium(EGM) with growth factors continuously for two months. We observed the adherence and morphology of cultured cells under a phase-contrast microscope, and detected the expression of CD31 through fluorescence-actived cell sorting(FACS),and examined the binding of DiI- labeled acetylated low-density lipoprotein(acLDL) , and detected the staining for the endothelial markers vWF expression on inverted fluoresecent microscope and biochemical assays for NO released from cultured cells.Results After 3 days culture,attached spindle-like cells Appeared and a number of cell clusters appeared within 7 days. Attaching cells expressed von Willebrand factorand CD31, uptook acetylated-LDL and released nitric oxide. The adherent cells approached confluence after 10 passages, and increased about 210~310 times in number. Conclusion A subset of adult BM-MNC could differentiate into cells with phenotypic characteristics of endothelium under the habitat for EC in vitro.The culture have favourable shape and founction ,and increases in stage number,which offers possibility in transplantation...