The Effect Of Osteopontin On Mesenchymal Stem Cell Directed Migration And Its Molecular Mechanism

Mesenchymal stem cells (MSCs) exhibit the potential to give rise to cells of diverse lineages and are characterized by their capacity to differentiate into multiple mesenchymal cell lineages under various culture conditions. The use of MSCs for therapeutic applications has been hailed with the increasing evidences. Besides the advantages of efficient isolation and culture, in vitro huge proliferation potential, non-involvement in medical ethics, MSCs are inclined to migrate to sites of tissue injury when injected intravenously. The ability of directed migration into injury sites is the key of MSCs clinical application.Osteopontin (OPN) is synthesized in a variety of tissues and cells. It is up-regulated in response to injury in every organ examined. The increased expression of OPN has been associated with increased cell mobilization and directed migration of cells. Therefore, the facts imply that OPN may contribute to the recruitment of MSCs to the sites of injury.The role of OPN on MSCs directed migration and the molecular mechanisms have not been studied well. Thus, this thesis tried to figure out the role of recombinant OPN on the directed migration of rat MSCs (rMSCs) and its molecular mechanisms.The research contents and results are listed as follows:1. rMSCs isolation and characterization rMSCs were obtained by density gradient centrifugation with 1.073 g/ml Percoll. The isolated cells achieved 85% confluence within 14 days. Adherent cells appeared after cells had been passaged within half an hour and spread thoroughly within 24 h and showed fibroblast-like morphology. Passaged cells achieved 80~90% confluence within 4~6 days and exhibited circinate orientation when achieving confluence. Flow cytometry showed that the surface antigen CD44 and CD90 were positive while CD34 was negative.2. OPN induces rMSCs directed migrationThe directed migration promoting activity of OPN in rMSCs was examined by transwell migration assay. OPN stimulated rMSCs migration without changing the number of viable cells in a concentration-dependent manner.3. rMSCs require integrinβ1 for directed migration induced by OPNTo determine whether the expression of CD44v6 or integrinβ1 mRNA was influenced by OPN stimulation during the migration process which might contribute to OPN-induced rMSCs migration, rMSCs were subjected to OPN (1μg/ml) and the mRNA was detected by RT-PCR. The mRNA expression of integrinβ1 increased (1.5 folds, p<0.01). However, OPN did not change the mRNA expression of CD44v6. In addition, western blot showed that integrinβ1 protein increased at 2.0 h and peaked at 4.0 h, which was about 1.3 folds (p<0.05) higher than that of control group. After 6.0 h of stimulation of OPN, the expression of integrinβ1 protein was decreased while still at a higher level compared with that of control group though no significant difference was detected. Blockade of integrinβ1 could inhibit rMSCs migrating towards OPN.4. Focal adhesion kinase (FAK)/ extracellular signal regulated kinase (ERK) signaling pathways are responsible for OPN-induced rMSCs directed migration.Western blot was used to detect the expression of phospho-FAK (pFAK) and phospho-ERK1/2 (pERK1/2) after OPN (1μg/ml) stimulation. The results showed that pFAK increased at 0.5 h (1.3 folds, p<0.05) while pERK1/2 increased at 0.5 h (1.7 folds, p<0.05), peaked at 1.0 h (2.8 folds, p<0.01) and decreased at 2.0 h (1.3 folds, p<0.05). After 4.0 h, there was no difference compared with control groups. Inhibitors of FAK and ERK could block the rMSCs directed migration induced by OPN. Additionally, integrinβ1 antibody could inhibit the OPN-induced elevated expression of pFAK and pERK1/2.5. OPN changes the mechanical character of rMSCsAtomic force microscope (AFM) was used to detect young's modulus of rMSCs stimulated by OPN (1μg/ml). OPN could decrease young's modulus significantly (p<0.05). In addition, blockade of integrinβ1 or activation of FAK/ERK inhibited the decrease in young's modulus induced by OPN. The results represented a close connection between rMSCs stiffness and migrationOur results showed that OPN could increase mRNA and protein expression of integrinβ1, then activate FAK/ERK signaling pathways and lessen the cell stiffness of rMSCs, and finally induce rMSCs directed migration. The up-regulation of OPN in response to injury and inflammation in vivo may induce the migration of MSCs into the sites of injury and contribute to tissue repairs.