| Part one Isolation, culture and biological characteristics of rat umbilical mesenchymal stem cellsObjectiveTo explore the isolation, culture and purification of rat umbilical mesenchymal stem cells (UCMSC) and investigate the biological characteristics.MethodsMesenchymal stem cells were isolated from rat umbilical cord (UC) on 16th day of pregnancy by collagenase and trypsin. The morphological of cells was observed. Cell proliferation was detected by MTT assay. Cell surface markers of CD 34, CD 45, CD 90, CD l05, CD l06 of UCMSC were analyzed by flow cytometry and immunocytochemical method. Differentiate potency of UCMSC to osteogenic cells and adipocytes were evaluated.ResultsUMSCs were harvested successfully from UC by collagenase and trypsin. Adherent cells were observed after 24 hours. As the passage number increased, cells appeared as fusiform. UMSC proliferated quickly, double time of (32.37±1.33) h . Flow cytometric analysis and immunocytochemical method showed that the UMSCs expressed CD 90, CD 105 and CD 106, but did not express CD34 and CD45. Cell cycle analysis showed that 87.12%±1.32%cells were in Go/Gl phase. UMSCs have the ability to differentiate into the osteogenic cells and adipocytes. ConclusionThe trypsin and collagenase digestion procedure is a easy, convenient and reliable method to obtain UMSCs. The UMSCs have the same immunophenotype with bone marrow mesenchymal stem cells and human UMSCs. The cells have strong proliferation capacity and the multiplex differentiation potential.Part two Study on the expression of CXCR4 on umbilical cord mesenchymal stem cells and its influencing factorsObjectiveTo observe the expression of CXCR4 on UMSCs and evaluate the effect of IL-2, IL-6, LPS, LFS, NS, CM (include IL-2, IL-6, LPS, SCF, TNF-α), SCF and TNF-αon CXCR4 expression.MethodsThe 3rd generation of UMSCs at 70%~80% fusion were randomly assigned to 9 groups: control group(normal culture medium), IL-2 group(IL-2 20ng/mL), IL-6 group(IL-6 20ng/mL), LPS group(LPS 20ng/mL), LFS group(LFS 20ng/mL), NS group(10% normal rat serum), CM group(IL-2 20ng/mL, IL-6 20ng/mL, LPS 10μg/mL, SCF 50ng/mL and TNF-α20ng/mL), SCF group (SCF 20ng/mL) and TNF-αgroup(TNF-α20ng/mL). The expression of CXCR4 was assessed by RT-PCR, immunocytochemistry, western blot and flow cytometry after 24hs.ResultsThe expressions of CXCR4 on the surface of UMSCs in groups stimulated with IL-2, IL-6, LPS, LFS, CM, SCF and TNF-αwere 27.83%±1.89%, 22.40%±1.81%, 19.07%±1.17%, 42.37%±1.60%, 40.17%±1.78%, 31.4%±1.61% and 16.37%±1.75% respectively, which had statistical difference compared with CON group(9.67%±1.06%) (P<0.05). The expression of CXCR4 in NS group was 9.7%±1.3%, with no statistical difference compared with control group (P > 0.05). RT-PCR,western blot and immunocytochemistry method showed that IL-2, IL-6, LPS, LFS, CM, SCF and TNF-αcould stimulated CXCR4 mRNA transcription and CXCR4 expression (P < 0.05, compared with control group). But those in NS group and CON group were similar (P>0.05).ConclusionsCXCR4 was expressed on both cytomembrane and in cytoplasm of mesenchymal stem cells.Blood serum from liver failure rat could induce high expression of CXCR4, which might be associated with IL-2, IL-6, LPS, SCF and TNF-α. |
PDF Full Text Request