| Objective:1. To isolate the mesenchymal stem cells (MSCs) from bone marrow and peripheral blood of burned and nromal rats, and to compare the biological behavior of MSCs from different sources. 2. To investigate the effects of burned rat serum, tumor necrosis factor-a (TNF-a), granulocyte-colony stimulating factor (G-CSF) on the expressions of ICAM-1 and VCAM-1 and on migration ability and adhesive function of MSCs. 3. To explore the regulation roles of JAK-STAT,MAPK and NF-κB signaling pathways in the migration ability of MSCs.Methods: 1. The MSCs were isolated from bone marrow and peripheral blood of burned and normal rats. Immunocytochemistry, Western blotting and flow cytometry analyses were used to characterize these isolated MSCs. The proliferation ability and biological characteriscs were also compared among the MSCs from different sources. 2. Immunocytochemistry, Western blotting and RT-PCR were used to observe the effects of burned rat serum, TNF-a and G-CSF on the expressions of intercellular adhesion molecule(ICAM)-l and vascular cell adhesion molecule(VCAM)-1 in MSCs. Chemotactic effects of MSCs affected by the three factors were investigated by using transwell system. Effects of these three factors on adhesion ability in MSCs were also observed through the method of Matrigel adhesion. The correlation action of TNF-a and G-CSF were identified through the neutralization experimentation of antibodies for TNF-a or G-CSF. 3. The activation of intrarcellular NF-κB in MSCs after being treated with TNF-a was tested by electrophoretic mobility shift assay (EMSA). Western blotting analysis was used to observe the activation of JAK-STAT and MAPK signaling pathways. The effect of TNF-a on the migration ability and expression of ICAM-1 of MSCs was also observed after signaling pathways had been interupted by usingthe inhibitor of ERK (PD98059) or the inhibitor of p38 (SB203580).Results: 1. MSCs could be isolated from peripheral blood of some burned rats, but not from peripheral blood of normal rats. The morphology and phenotype of MSCs from peripheral blood of burned rats were consistent with those from rat bone marrow. The growth curves were similar among different cell groups. Clone-forming ability of MSCs derived from peripheral blood was lower than that of other groups. 2. the expression of ICAM-1 could be up-regulated by burned rat serum and certain concentrations of TNF-a, whereas the expression of VCAM-1 remained unchanged during the process. The treatment of G-CSF had no effect on the expressions of ICAM-1 and VCAM-1. At the same time, burned rat serum and a certain concentration of TNF-a could enhance migration ability and adhesion function of MSCs. Certain concentration of G-CSF could affect the adhesion function of MSCs, but not the migration ability. Neutralization experiments showed that the effects of TNF-a and G-CSF could be suppressed by their neutralization antibodies respectively. 3. After being treated with certain concentration of TNF-a, the expressions of phospho-ERK and phospho-p38 were increased. SB203580 could suppress the up-regulation effect and chemotactic effect of MSCs induced by TNF-a. PD98059 had no such effects as SB203580.Conclusion: 1. The MSCs could migrate from bone marrow to peripheral blood after the rats were burned. 2. Treatments of burned rat serum or TNF-a could up-regulate the expression of ICAM-1 and enhance migratiom ability and adhesion function of MSCs. 3. The enhancement of migration ability induced by TNF-a might be associated with the up-regulation of ICAM-1 expressed by MSCs. 4. p38 signaling pathway might be involved in the change of migration ability induced by TNF-a. 5. The study provide the experimental foundation of the clinical application of autogeneic stem cells to participate wound repair.
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