The Effect Of Mechano-growth Factor On Proliferation And Migration Of Bone Marrow-derived Mesenchymal Stem Cells And Its Involved Mechanisms

Mesenchymal stem cells (MSCs) are a kind of special stem cells with self-renewaland multi-lineage differentiation potentials. Under certain environmental conditions,MSCs can directionally differentiate into various cell types, such as osteoblasts,chondrocytes, adipocytes, muscle cells, nerve cells, etc. Numerous studies havedemonstrated the positive role of MSCs in tissue repair and regeneration. It is one of thekey parts for function of tissue repair of MSCs to mobilize from bone marrow andmigrate through peripheral circulation into injured tissues. The researches in recentyears have proved that various mechanical and chemical factors play a significant partin regulating the directed migration of MSCs to the damaged tissue.Mechano-growth factor (MGF) is an alternative splice variant of the igf-I gene,expressing in various tissues/cells and having the force sensitivity. Studies confirmedthat MGF can activate satellite cells, promote myoblast proliferation, and play animportant role in treatment of muscle defects, prevention of myocardial damage andrepair of damaged nerve.Currently, the effect of MGF on the biology behavior of MSCs is still lack ofcomprehensive understanding, even we know very little about the molecular mechanisminvolved in this process. Therefore, this article uses a synthetic MGF carboxy-terminal25amino acids of rat’s E peptide (MGF-C25E) to examine the effect of MGF-C25E onproliferation and migration behavior characteristics and related mechanism of rat MSCs(rat MSCs, rMSCs).The main content and the results of this study are as follows:1) Cultivation and identification of rat mesenchymal stem cells (rMSCs)Gradient centrifugation and attachment culture were used for rMSCs isolation. Cellcultured and passaged with DMEM containing10%fetal bovine serum. Microscopicobservated cell morphology and flow cytometry checked the expression of cellsurface-associated antigen and the distribution of cell cycle. Results showed thatadherent fibroblast-like morphology cells appeared after12-24hours and these cellswere almostly covered after7-10days. Separated rMSCs in this way showed swirldistribution, spindle shaped and tended to be more consistent. The cells in the growthprocess can be fully spread out without laminated growth phenomenon. Further cellsurface markers and cell cycle detection elaborated that CD34negative, CD90positive,CD44positive, which was in keeping with the general characteristics of the surface markers of MSCs, and94.23±0.84%of rMSCs were in G0/G1phase, which showedmost of the cells were in stationery stage, according with the characteristics of stemcells period distribution.2) The effect of MGF-C25E on proliferation of rMSCsMGF-C25E is a synthetic mechano-growth factor E peptide containing25aminoacids. To investigate the proliferation of rMSCs stimulated by MGF-C25E, MTTassay and cell counting assay were performed. The results showed that MGF-C25E atconcentrations of10-70ng/mL had no impact on the proliferation of rMSCs within24-96h compared with the control. The results suggested that MGF-C25E did notsignificantly affecte rMSCs proliferation under the conditions of the concentrations andtime.3) The effect of MGF-C25E on migration of rMSCsThe migration of rMSCs in response to MGF-C25E in vitro was examined bytranswell assay. A significant increase of migratory cell number of rMSCs was observedin the presence of all case of MGF-C25E treatment compared with the0ng/mL(control),and peaked at50ng/mL and then gradually declined as MGF-C25E concentrationincreased. The results confirmed that MGF-C25E can significantly promote rMSCsmigration.4) Molecular mechanism of MGF-C25E promote rMSCs migrationRT-PCR assay found that MGF-C25E also enhanced the expression of SDF-1geneand CXCR4gene in rMSCs. Western blot confirmed the upregulation of CXCR4inresponse to MGF-C25E (p<0.05). AMD3100, the inhibitor of CXCR4, couldsignificantly inhibit MGF-C25E-induced phosphorylation of ERK1/2in rMSCs as wellas the enhanced migration.rMSCs were then subjected to50ng/mL MGF-C25E and the expressions ofphosphorylation ERK1/2(p-ERK1/2) were detected by western blot from0min to90min and normalized to total ERK1/2(t-ERK1/2), respectively. After MGF-C25Estimulation, expressions of p-ERK1/2were significantly increased and reached a peakat60min (p<0.05). However, the MGF-C25E-induced expression of p-ERK1/2inrMSCs was significantly decreased after treatment with MEK/ERK1/2inhibitorPD98059as well as the enhanced migration which indicated that MGF-C25E promotedrMSCs migration by activation of ERK1/2. These results demonstrated that50ng/mLMGF-C25E could promote rMSCs migration, and SDF-1/CXCR4-ERK1/2signalpathway may play a crucial role in this process. 5) The mechanical mechanism of MGF-C25E promotes rMSCs migrationChange in cell stiffness, which is characterized by Young’s modulus, has beenfound to be coupled with change in cell-migratory behavior.We assessed the stiffness ofrMSCs under the simulation of MGF-C25E(50ng/mL) for12h with or withoutAMD3100(50μg/mL), PD98059(50μM) by AFM. The data shown that cells withMGF-C25E were significantly stiffer than the control, while inhibitors of CXCR4orERK1/2could restore the increase in Young’s modulus induced by MGF-C25E. Cellstiffness is determined by the structure of cytoskeleton. In this study, we sought todetermine whether MGF-C25E could increase cell stiffness by changing actincytoskeletal structure.The images showed that rMSCs, stimulated by MGF-C25E for12h, had more well-defined stress fibers than the control and hence exhibited aintensified actin filamentous network. Inhibitors of CXCR4or ERK1/2also preventedMGF-C25E-induced cytoskeleton change. These results indicate that, MGF-C25E maypromote F-actin polymerization and increase the hardness throughSDF-1/CXCR4-ERK1/2signaling pathway, and ultimately promote cell migrationbehavior of rMSCs.