| Background and ObjectiveBasic fibroblast growth factor (bFGF) is a mitogenic heparin-binding protein, has a more extensive biological activity. bFGF can induce a variety of cell proliferation and differentiation and has an important effect in blood vessel growth, wound healing, tissue regeneration and repair, nerve regeneration and so on. bFGF has been more widely used in nerve regeneration studies, including basic research and clinical application. Literature shows that bFGF can induce human umbilical cord blood mononuclear cells to a variety of nerve cell differentiation, the cells transplanted to the nerve injury models, repaired of nerve is very significant. Because bFGF has pleiotropic biological activites and the broad spectrum of neurotrophic and recombinant human bFGF has also appeared bFGF revealed a bright prospect for clinical application. But so far, bFGF induced differentiation of umbilical cord blood mononuclear cells mechanism is not clear, in order to explore the effect of bFGF on the different stages of hematopoietic stem/progenitor cell differentiaton and promote cord blood mononuclear cells induced by bFGF clinical application,this study using geng chip technology, analysis of gene expression of cord blood CD34+and CD133+cells differentiation induced by bFGF and SCF in vitro, so as to investigate mechanisms of bFGF-induced cord blood CD34+and CD133+cells differentiation to provide theoretical support and experimental data and lay the foundation for clinical application.MethodsHuman umbilical cord blood CD34+and CD133+cells were isolated and purified by MACS magnetic beads selection. Selected rate of CD34+and CD133+ hematopoietic stem cells was detected using flow cytometry; The CD34+and CD133+ cells were cultured for 10 to 15 days in DMEM/F12 medium, supplemented with SCF, bFGF and B27, CD34+and CD133+cell morphological changes were measured before and after bFGF induction; Total RNA from these cells was extracted and the concentration and purity of RNA were determined by agarose gel electrophoresis degeneration; The genetic level of these cells was performed using Oligo GEArray(?) chip and GEArray software.Results(1) Selected rate and purity sorted of CD34+and CD133+:The 20 samples of cord blood were isolated and purified respectively, CD34+cell purity (77.52±5.06)%, recovery rate(2.74±1.59)%; CD133+cell purity(79.16±3.37)% and the recovery (1.12±0.94)%.Repeat magnetic separation step increase the purity of the cells to surpass 90%; Rate of living cells for more than 95% by trypan blue staining.(2) The morphological of CD34+cells and CD133+cells before and after the culture: The new selected CD34+cells and CD133+cells were spherical, Following induced by bFGF and SCF for 15 days, cells were significantly amplified to 2-3X, the majority of cell morphology did not find significant changes, some cells changed into an irregular shape and were adherent growth.(3) Genetic changes before and after culture of the CD34+cells:In the detection of 263 genes related to stem cells, a twofold up-regulation of 10 genes was detected in SCF and bFGF-induced CD34+cells, whereas 20 genes showed a down-regulation compared with fresh-separated CD34+cells; Downregulated expression of hematopoietic cell lines markers of CD19 and CD3D; upregulated expression of mesenchymal cell lines markers of PPARG and SPP1, as well as neural cell lines markers of S100B in cell differentiation markers. The resultes shows that the growing trend of differentiation to mesenchymal cells and neural cells of CD34+cells induced by bFGF.(4) Genetic changes before and after culture of the CD133+cells:In the detection of 263 genes related to stem cells, a twofold up-regulation of 21 genes was detected in bFGF-induced CD133+cells, whereas 7 genes showed a down-regulation compared with fresh-separated CD133+cells. These genes were involved in stem cell specific markers, cell cycle regulators, stem cell differentiation markers and signaling pathways that are important for stem cell maintenance. Blood cells symbol CD3D and CD247 expression decreased and CD4 expression increased after the culture, upregulated expression of mesenchymal cell lines markers of PPARG and SPP1 and neural cell lines markers of S100B after the culture. The resultes shows that the growing trend of differentiation to mesenchymal cells and neural cells of CD133+cells induced by bFGF.(5) In the detection of 263 genes related to stem cells, two-fold difference of 10 genes in umbilical cord blood CD34+cells and CD133+cells. After bFGF-induced culture,32 kinds of gene expression of CD133+cells were significantly higher than CD34+.There are no gene lower than CD34+among detected 263 genes.ConclusionIn the detection of 263 genes related to stem cells, there are only a few gene expression differences of cord blood CD34+and CD133+cells; The reaction of CD133+cells on bFGF are significantly stronger than CD34+cells; CD34+cells and CD133+cells induced by bFGF and SCF have strong differentiation trend to mesenchymal cells and neural cells.
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