POSTN Regulates The Self-renewal Ability Of Placental Stem Cells And Its Effect On Wound Healing

Refractory wounds caused by diabetes remain an unsolved problem,bringing huge mental and economic burden to patients.Mesenchymal stem cells have multiple differentiation potential and can promote diabetic wound healing,but the self-replicating aging of stem cells will affect the therapeutic effect of stem cells.Our previous study found that among the differentially expressed genes of young human placental stem cells（pla MSCs）and aging pla MSCs,the expression of POSTN was significantly down-regulated in aging pla MSCs,suggesting that POSTN may be related to the self-renewal ability of pla MSCs.Objective:To study the effect of POSTN gene on the self-renewal ability of pla MSCs and the effect POSTN-overexpressed pla MSCs on wound healing.Methods:The expression of POSTN in senescent and young pla MSCs was detected.After knock-down or overexpressing the POSTN gene of pla MSCs,detected the senescence relatedβ-galactosidase activity and the expression of p16 and p21 by western blot.To investigate whether POSTN regulates the senescence of pla MSCs,the cell activity at day 1,3,5,7 and 9 was measured by MTT assay to understand the cell proliferation ability,and the cell cycle was detected by flow cytometry.The three-line differentiation experiment and the expression of stemness markers NANOG and OCT4 was detected by RT-qPCR to study whether POSTN affected the differentiation and stemness of pla MSCs.In animal experiments,the wound model of diabetic rats was established.Pla MSCs with overexpression of POSTN(POSTN^+/+MSCs),pla MSCs transfected with negative plasmid（veh MSCs）or pla MSCs group without treatment（pla MSCs）were locally injected into the wound,and PBS injection group was used as blank control group.The wound healing was observed.HE staining was used to detect the migration degree of epithelial cells in the wound tissue.Masson staining was used to detect collagen deposition,and real-time PCR was used to detect the typeⅠ/Ⅲcollagen fiber ratio.Results:We found that POSTN expression was down-regulated in senescent pla MSCs,and knockdown POSTN causes the pla MSCs to accelerate aging,such as the increase ofβ-galactosidase activity,increased the expression of p16 and p21.In addition,the cell activity of pla MSCs after knockdown POSTN was significantly weaker than the control group pla MSCs on day 5,7 and 9.Pla MSCs knock-downed POSTN also showed a decreased proportion of S-phase cells and the expression of Akt,accompanied by osteogenic and chondrogenic differentiation ability and stemness damage.However,overexpression of POSTN could alleviate the aging of pla MSCs.On the 7th day of proliferation experiment,the cell activity was significantly stronger than that of the control group.In cell cycle experiment,the proportion of pla MSCs in S phase increased,the proportion of pla MSCs in G2/M phase decreased.The expression of Akt in pla MSCs overexpressing POSTN was increased,and the osteogenic and chondrogenic differentiation ability and stemness of pla MSCs were improved.In animal experiments,POSTN^+/+MSCs showed a stronger ability to promote wound healing than inartificial pla MSCs and veh MSCs,mainly by promoting wound contraction.However,POSTN^+/+MSCs did not show a stronger role in promoting re-epithelialization and collagen deposition,and the type I/III collagen fiber ratio was not statistically different from the other two treatment groups.Conclusion:POSTN regulates the self-renewal ability of pla MSCs through the AKT pathway,and the expression of POSTN is positively correlated with the growth ability of pla MSCs.Overexpression of POSTN can improve the healing effect of pla MSCs on diabetic chronic wounds by enhancing pla MSCs to promote wound contraction.This study provides a new approach for the application of stem cells in the treatment of chronic refractory wounds caused by diabetes mellitus.