| Objective To observe the basically biological characteristics of human umbilical cord blood mesenchymal stem cells (hUCB-MSCs), which were cultured using the two culture media sequential cultural system. Methods The mononuclear cells were isolated by density gradient centrifugation from human umbilical cord blood, and sequential cultured in DMEM/F12 medium containing 10% fetal bovine serunl and human umbilical cord mesenchymal stem cell growth medium (OHUCMSCG medium). The cell maker proteins were determined by immunocytochemistry and flow cytometry methods. Then the cell growth curve was draw by determine of cell proliferation using MTT methods. The differentiation potential were evaluated for osteogenic and adipogenic differentiation by alizarin red and oil red staining. Results The colony forming rate of hUCB-MSCs was high in DMEM/F12 original culture compared with in MesencultTM mesenchymal stem cell specific medium, and subcultured to passage 2nd using OHUCMSCG medium. It was benefit for purifying hUCB-MSCs by differential adhesion. Then hUCB-MSCs were sequentially cultured in OHUCMSCG medium from passage 3rd to 8th. hUCB-MSCs all showed spindle-shaped fibroblasts- liked cells, and arranged showing whirlpool-shape, radiated shape. These cells expressed CD29+, CD44+, CD73+ CD166+ and vimentin, but expression of CD34 and CD45 were negative. Growth and proliferation of passage 6th hUCB-MSCs were better. The double proliferation time was 68.1 1h. The log exponential stage was from 2d to 6d. After the cells were cultured 21 days in fit condition, hUCB-MSCs were induced to positive osteogenic and adipogenic cells of alizarin red staining mineral extracellular matrix and oil red staining neutral lipid vacuole, respectively. Conclusion hUCB-MSCs could be successfully expanded in DMEM/F12 and OHUCMSCG medium by sequential culture. The basic characteristics of mesenchymal stem cell were keeped perfectly in this culture system. Objectives To observe Ginsenoside Rgl (Rgl) on the proliferation of human umbilical cord blood mesenchymal stem cells(hUCB-MSCs) and investigate the underlying mechanism by considering calcium-sensing receptor (CaSR) signaling as the target point. Methods:After five or six consecutive passages, the hUCB-MSCs were randomly allocated into eight groups:Rgl low to high five dose group, Rgl+blocking agent CdCl2 and CdCl2 group. the proliferation of hUCB-MSCs was assayed using MTT method. Cytokine in culture supernatant were detected by ELISA assay. Real time quantitative polymerase chain reaction (RT-PCR) was used to evaluate hUCB-MSCs CaSR mRNA expression. CaSR, p-p38 and p-ERK protein expressions were detected using immunofluorescence and Western blot. Result:Compared with control group, the proliferation of hUCB-MSC in all Rgl treatment groups has no significant difference(P>0.05), but CdCl2 groups could obviously inhibit the growth of hUCB-MSC(P<0.05), and the inhibitory effect was not reversed by treatment with Rgl. hUCB-MSC expressed IL-8,IL-6,LIF and TGF-β1, low expressed or not the other cytokines IL-7, IL-12, SCF, GM-CSF and M-CSF. Rg1 don't affect the content of above cytokines compared with control group. The expression of CaSR mRNA was low in control group. Compared with control group, the expression of CaSR mRNA were no difference among 1μmol/L,3μmol/L Rgl group and CdCl2 group(P>0.05). But Rgl 3μmol/L and CdCl20.01 mmol/L+Rgl 1 u mol/L group could inhibit p-p38 and p-ERK protein expressions.The expression of CaSR protein was no significant change in Rgl group compared with control. Conclusion:Rgl has no influence on proliferation and the CaSR protein expression of hUCB-MSCs, but it could down regulation the expression of phosphorylating protein ERK and p38. hUCB-MSCs could secret IL-8, IL-6, LIF and TGF-β1, but didn't or slightly release IL-7, IL-12, SCF, GM-CSF and M-CSF. These results suggested that Rg1 maybe affect other biological characteristic of hUCB-MSCs by downregulate activity of ERK and p38. |
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