Effects Of PPP And PRP On Biological Characteristics Of BM-MSCs In Vitro

Objective:Aimed to investigate the effects of platelet-poor plasma(PPP)and platelet-rich plasma(PRP)on the biological characteristics of bone marrow-derived mesenchymal stem cells(BM-MSCs).In order to improve the relationship between BM-MSCs and platelet plasma,and provide some theoretical basis for the rational use of platelet plasma in clinical diseases.Methods:(1)Using the adopted tissue adherent to isolate and culture the BM-MSCs.Then,we identified the morphological characteristics,differentiation potential and cell surface antigen of BM-MSCs;(2)MTT assay was used to detect the effect of PPP and PRP on the proliferation of BM-MSCs.We chose 100×10~6/ml and 48h as the most suitable concentration and time for the experiment;(3)The migration ability of BM-MSCs was tested by transwell migration assay and scratch assay.Then,we detected the senescence-associated?-galactosidase staining and cell colony formation assay after pre-treated with PPP and PRP;(4)The cell cycle and cell apoptosis were analyzed by flow cytometry;the multi-differentiation capacities and DAPI staining of BM-MSCs were also evaluated;(5)The expressions of related proteins were tested by immunofluorescence and Western blot.Results:(1)After isolated from bone marrow and cultured for up to 14 days,the cells were displayed a polygonal,spindly and fibroblast-like morphology.According to flow cytometric analysis,the BM-MSCs were positive for CD29,CD44 and CD90,but negative for CD19,CD34,CD45,CD133 and HLA-DR.Afterwards,the cells were induced to undergo adipogenic or osteogenic differentiation and stained with Oil Red O or ALP;(2)The MTT assay showed that PRP could promote cell proliferation to a certain extent and the effect of PPP is not obvious;(3)The migration ability of BM-MSCs was detected by scratch assay and transwell migration assay.Our results indicated that PRP has a positive effect on the migration ability of BM-MSCs.The?-galactosidase staining showed that PRP could significantly improve cell aging.Next,the cell colony formation assay demonstrated that PRP can enhance cell cloning ability;(4)The multi-differentiation capacities of BM-MSCs revealed that PRP had no significant effect on adipogenic potential of BM-MSCs,but PRP can enhance the osteogenic potential of BM-MSCs when they were co-cultured with PRP.We do not find obvious difference from the results of cell cycle assay and apoptosis assay;(5)From the immunofluorescence detection and Western blot analysis,we found that the expression of EMT related proteins was changed and several pluripotency marker genes for instance Sox2,Sall4,Oct4 and Nanog were also had varing degrees of increased.In subsequent studies,the expression levels of main factors in the phosphoinositide 3-kinase(PI3K)/protein kinase B(AKT)signal pathway were examined.Our findings suggested that PRP could promote cell migration of BM-MSCs by activating the PI3K/Akt signaling pathway.Conclusions:(1)In our study,we speculated that PRP in low concentrations(PLT?100×10~6/ml)could significantly influence the cell proliferation,cell migration,the osteogenic differentiation capacities and the expressions of related proteins;(2)nder the culture condition in vitro,we didn't find an obvious effect on cell cycle,cell apoptosis and the adipogenic differentiation capacities of BM-MSCs after pre-treated with PPP and PRP.The above results showed that the enrichment of platelet may have a certain effect on BM-MSCs in inflammation or injury sites,which is beneficial to the elimination of inflammation and repair of injuries.