| Mesenchymal stem cell is one member of the adult stem cells. Most of the mesenchymal stem cells which under research now are derived from adult tissue of human or animals. In recent years, mesenchymal stem cell from umbilical cord has becoming a research focus by reason that it is abundant in source, ealy to obtain, and has no adverse effects on donors or any restrictions on ethic. In addition, it has been reported that vitamin A plays an important role in promoting the proliferation of various cells in vitro. In the experiment, we obtained mesenchymal stem cells from human umbilical cord, and identified them by immunohistochemistry and RT-PCR. We then studied the effect of vitamin A on these cells by means of detecting the proliferation of the detached cells, proliferational genes, and apoptotic genes. Simultaneously, we also tested the expression and secretion of the factors participating in cell proliferation with RT-PCR and Elisa, in order to explore its biological characteristics. The results obtained are as follows:1.Separation and identification of mesenchymal stem cells from human umbilical cordSeparating the umbilical cord cells by enzymic digestion, and the results showed that (1) the optimal strategy to digest the cells was that the cells were treated with a composite enzymes including 0.1%DNASE,0.1%Ⅵ collagenase, and 0.1% hyaluronidase at 4℃ for overnight, and then cultured at 37℃ for 25min with the presence of 0.25% trypsin and 0.02%EDTA; (2) human umbilical cord tissue cells became round or oval in shape after enzymatic digestion, and suspended in culture solution. After culturing for 1-2 days, the cells grew with adherence completely, and became fibroblast-like in shape; (3) a sigmoid growth curve of the mesenchymal stem cells was first tested. The phase of logarithmic growth appeared 2 days latter, and they hit a plateau in another 6 days; (4) The survival rate of the cells before frozen was 90.23%, and the survival rate of the postthawed cells was 87.61%; (5) immunohistochemistry revealed it that these cells did express the marker gene of mesenchymal stem cells CD29, CD44 and CD105, and the marker gene of stem cells Oct4, Nanog and Sox2 but the marker gene of epithelial cell CD31 nor the marker gene of hematopoietic stem cell CD34.All the results above confirmed that the cells obtained was mesenchymal stem cells, and conformed to requirements further test.2. The effect of vitamin A on the growth of human umbilical cord mesenchymal stem cellsWe analysed the effect of vitamin A on the growth of human umbilical cord mesenchymal stem cells by adding vitamin A with a concentration of 1 μmol/L into FBS+DMEM/F12 culture medium and KSR+DMEM/F12 culture medium, respectively. The results showed that (1) the MTT test showed that after adding vitamin A, the cells grew faster; (2) after RT-PCR, we found that in the cells treated with vitamin A, the expression of the genetic markers of blood stem cells, Nanog, and the proliferational gene, C-myc, increased, well the expression of the apoptotic gene, Bcl-x, reduced; (3) the results of immunohistochemistry and RT-PCR showed that culture the cells for 20 generations, they still expressed the marker gene of mesenchymal stem cells CD29, CD44 and CD105, and the marker gene of stem cells Oct4, Nanog and Sox2 but the marker gene of epithelial cell CD31 nor the marker gene of hematopoietic stem cell CD34.All the results above confirmed that 1μmol/L vitamin A may up-regulate the expression of Nanog gene, activating cell proliferation signal transduction pathway, and regulat the expression of hyperplasia gene PCNA, C-myc as well as cell apoptosis-related gene Bcl-x, which promoted the proliferation of the human umbilical cord mesenchymal stem cells.3. The effect of vitamin A on the secretion of factors from human umbilical cord mesenchymal stem cellsWe tested the expression and secretion of cell growth factors of 4 groups of cells which were cultured in FBS+DMEM/F12 culture medium or KSR+DMEM/F12 culture medium that both contained 1 μmol/L vitamin A at different time points. The results showed that (1) the expression of factors LIF, SCF, CSF1, EGF and bFGF was detected by RT-PCR in human umbilical cord mesenchymal stem cells.The expression of gene EGF was higher in cells grew in cultures in presence of KSR than that of FBS. Only the expression of gene SCF increased after adding vitamin A into the cultures. It reveals that KSR increases the expression of EGF, and vitamin A promotes the expression of SCF. (2) human umbilical cord mesenchymal stem cells secreted SCF, CSF1 and EGF, but bFGF or LIF. The secretion of EGF was exocytosis for its expression in culture was higher than in cells. Besides, the addition of vitamin A had little effect on the secretion of the other five cell growth factors. |
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