| Objective:Mesenchymal stem cells(MSCs)have been widely applied in regenerative medicine and clinical treatment of various diseases due to their great ability in proliferation,differentiation and immunomodulatory regulation.However,MSCs will cause repetitive aging with the increase of passage.The ability of proliferation,differentiation and immunomodulatory regulation of ageing MSCs become weakened,which largely limits the their therapeutic efficiency and application.This study aims to develop novel strategies to intervene the MSC ageing during expansion.The secretion of PGE-2 as a measure marker of MSCs,trolox(6-Hydroxy-2,5,7,8-tetramethylbenzopyran-2-carboxylic acid)was identified as a stimulator of PGE-2 secretion by compound library screening experiments.Further studies explore its effect on the biological characteristics of MSCs,including aging and immunomodulatory ability.Methods:In the study,firstly,we observed the morphology of obtained young umbilical cord mesenchymal stem cells(UC-MSCs)under microscope,examined the differentiation characteristics,the surface markers(CD90,CD105,CD146,CD34,CD11 b,CD45),and the cytokine(PGE-2)secretion capacity.And then,we used β-galactosidase staining,flow cytometry,enzyme-linked immunosorbent assay,and RT-q PCR to compare the differences between young and aging MSCs.In order to explore the effect of trolox on the biological characteristics of MSCs,we first used CCK-8 assay and ELISA to detect PGE-2 secretion in this study to determine the optimal concentration of trolox treatment MSCs.Then,flow cytometry was used to detect the changes of MSCs surface markers after trolox treatment,and transwell co-culture of MSCs and macrophages after trolox treatment,so as to explore the effect of trolox treatment on the immunomodulatory ability of MSCs.In addition,RT-q PCR was used to detect the expression of senescence proteins P16 and P21 and the expression of mi R-17-92 clusters in MSCs after trolox treatment.In order to explore the mechanism of trolox on MSCs,we transferred mi R-17-92 clusters plasmids into MSCs and made them over-expression.In addition,A20 is the downstream target of mi R-17-92 clusters,so we verified the expression of A20 protein in MSCs after aging,MSCs after trolox treatment,and MSCs after over-expression mi R-17-92 clusters by western blot.In addition,since the tendency to aging of MSCs during in vitro culture may be related to oxidative stress,and trolox is also an antioxidant substance,in this study,we used colorimetric method to compare the activities of the most representative antioxidant enzymes,including catalase(CAT),glutathione peroxidase(GSH-PX)and superoxide dismutase(SOD),in cells after trolox treatment and detected the expression changes of prdx family.Results:Under microscopy,it can be observed that young UC-MSCs grow adherently vortex,small and narrow,and have good osteogenic as well as adipogenic differentiation ability,and the flow cytometry results showed that MSCs surface markers CD90,CD105,CD73 were positive,CD34,CD11 b,CD45 were negative,which met the standards of UC-MSCs surface markers,and young UC-MSCs had good PGE-2 secretion ability.In addition,the experiments confirmed that compared with young MSCs,the positive staining rate ofβ-galactosidase and the proportion of cells staying in the G0/G1 phase increased,and the differentiation ability of adipogenesis,osteogenesis and cartilage-forming differentiation capacity decreased down,and the secretion ability of the immunomodulator PGE-2 decreased.When trolox treated MSCs for 24 h and 48 h,the optimal concentrations of trolox were 0.25μM(P<0.0001)and 2.0 μM(P<0.0001),respectively,and the proliferation results showed that trolox concentrations exceeding 5 μM(P<0.01)could promote the proliferation of MSCs.In addition,the treatment of trolox did not alter the surface markers of MSCs and still showed CD34,CD45 negative,CD73,CD90,CD105 positive.After trolox treatment,the positive rate of MSCs β-galactosidase staining was lower and the expression of P16,a aging-related protein,was down-regulated,and the MSCs after trolox treatment could maintain more generations with the increase of passage.In addition,the co-culture of trolox-treated MSCs with macrophages promoted the differentiation of macrophages into anti-inflammatory M2 type macrophages and reduced phagocytic ability.After trolox treatment,the expression of mi R-17-92 clusters in MSCs is up-regulated and the expression of downstream target A20 is down-regulated.After the over-expression of mi R-17-92 clusters of MSCs,the β-galactosidase positive cells and A20 were down-regulated,and after co-culture with macrophages,the M2-type differentiation of macrophages was promoted and the phagocytic ability of macrophages was reduced.In addition,the results of colorimetric experiments showed that the activity of antioxidant enzymes CAT,SOD,GSH-PX and the expression of prdx5,prdx6,gpx4 genes of MSCs were up-regulated after trolox treatment.Conclusion:In summary,the study proves that trolox inhibits the aging of MSCs through antioxidant pathway and mi R-17-92 clusters-A20 pathway,thereby promoting the proliferation of MSCs,as well as promoting the secretion of immunomodulator PGE-2 and M2-type differentiation of macrophages and reducing macrophage phagocytosis to exert immunomodulatory effects.Therefore,the treatment of the antioxidant substance trolox may be beneficial for the in vitro culture of MSCs,thereby promoting their role in regenerative medicine and clinical treatment. |
PDF Full Text Request