Effect Of Rehmannia Glutinosa Polysaccharides (RPS) On The Differentiation Of Adipose Tissue-derived Mesenchymal Stem Cells (ADMSCs) Into Cardiomyocytes

ObjectivePluripotent cells have been identified in adipose tissue.Other groups have demonstrated in vivo differentiation of adipose tissue-derived mesenchymal stem cells(ADMSCs)into cells exhibiting biochemical and functional markers of cardiac myocytes,including spontaneous beating.Based on these observations, the aim of this research was to isolate ADMSCs from rat adipose tissue,and to study the effect of rehmannia glutinosa polysaccharides(RPS)on the biological characteristics of ADMSCs,and to observe the protective effects of RPS against adipose-derived mesenchymal stem cells(ADMSCs)injury induced by hydrogen peroxide(H₂O₂).Furthermore,detect the effect of RPS on the differentiation of ADMSCs into cardiacmyocytes.MethodsAccording to the aims mentioned above,the study was divided into there parts.First part Adipose tissue was obtained from male Wistar rat,and then treated with trypsinase and collagenase sequently.After centrifugated,ADMSCs was obtained,and then cultured in DMEM medium containing 10%FCS and passaged. Morphological changes of ADMSCs were visualized under reversal phase microscope.Immunocytochemistry was used to determine the surface markers CD13,CD31,CD44,CD45 and CD105.Treat ADMSCs with various concentrations of RPS.Then 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assay was used to detect the effect of RPS on the proliferation of ADMSCs.Second part ADMSCs was obtained in the first part and chose the third passage for use.Cultured ADMSCs was preincubated with RPS and oxidative stress injury was induced by H₂O₂.Then 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assay was used to determine the cell viability,and lactate dehydrogenase(LDH)contents were measured.Third part ADMSCs was obtained in the way mentioned above.Third-passaged ADMSCs were induced by 5-azacytidine and incubated for 24 hours.Then divide the cells into two groups.One group was treated with RPS,the other was the control group.And both groups of cells were cultured for 4 weeks serially.Then the induced cells were subjected to immunostaining for alpha actinin,and the differentiation rate of ADMSCs into cardiacmyocytes were detected through flow cytometry.ResultsImmunocytochemical staining showed that most of the obtained cells were CD13, CD44 and CD105 positive,CD31 and CD45 negative.After treated with RPS, ADMSCs had stronger ability of proliferation,and higher viability and appeared concentration-dependent.After H₂O₂ administration,ADMSCs viability was significantly reduced compared with control group(P＜0.01),while LDH leakage content were significantly higher than that in control group(P＜0.01). Preincubation with RPS at various concentrations remarkably inhibited the reduction of cell viability and the leakage of LDH.After treatment with 5-azacytidine,the cells increased in size and formed a ball-like appearance. Immunostaining of the induced cells for alpha actinin was positive.The induced ADMSCs that were treated with RPS had a higher differentiation rate into cardiacmyocytes than the control group.ConclusionRPS can promote the proliferation of ADM$Cs,and can protect ADMSCs from H₂O₂injury.In addition,ADMSCs can be chemically induced into cardiomyocytes,and RPS may increase the differentiation rate of ADMSCs into cardiacmyocytes.