The Therapeutic Effect Of CD73~+ Human Adipose Mesenchymal Stem Cells On Mice With Pressure Ulcers And Its Potential Mechanism

BackgroundPressure injury,also known as pressure ulcer,is a common clinical complication.Its high incidence and difficulty to cure make it a major problem in nursing work.Refractory pressure ulcers refer to stage 3 and 4 pressure ulcers.Due to ulceration of the skin,inflammation,and sinus formation,osteomyelitis and osteonecrosis are more likely to become clinical treatment difficulties.Cell therapy provides a new direction for refractory pressure ulcers currently.Mesenchymal stem cells(MSCs)have biological characteristics such as multi-directional differentiation,immunosuppression,secretion factors,and have become one of the most promising stem cells in the treatment of chronic wounds.MSCs is a heterogeneous cell population,composed of a variety of subpopulations with different biological characteristics,and it is of great clinical significance to find a dominant subpopulation for the treatment of refractory pressure ulcers.Adipose mesenchymal derived stem cells(ADMSCs)are widely used in clinical treatment because they are easy to expand and obtain in vitro,and have fewer ethical issues.CD73,an exogenous-5’-nucleotidase,is a glycoprotein on the cell membrane,with nucleoside enzyme activity and signal transduction function.At present,CD73 molecule has been used in the treatment of many diseases,such as tumor,myocardial injury,organ transplantation,and so on Evaluating the efficacy of CD73+ADMSCs in treating pressure ulcers and comprehensively analyzing its mechanism of action can provide theoretical basis for clinical development of CD73+ cells,and then provide molecular criteria for selecting cells for stem cell treatment of pressure ulcersObjectivesTo evaluate the efficacy of CD73+ ADMSCs in treating pressure ulcers in mice,and to analyze the mechanism of CD73 in ADMSCs promoting the repair of intractable pressure ulcersMethods1.Culture human adipose mesenchymal stem cells(ADMSCs)in vitro2.Interfering with the expression of CD73 in ADMSCs and inhibiting the activity of CD73 in ADMSCs.The GFP-labeled CD73 interference lentiviral vector was constructed,and ADMSCs were transfected to inhibit CD73 expression.Western Blotting and Realtime PCR were used to detect the inhibitory effect.APCP was used to inhibit CD73 activity3.Inoculate ADMSCs into a 6-well plate,add substrate AMP,and experiment groups culture control group,ADMSCs group,ADMSCs+AMP group,ADMSCs+APCP+AMP group,ADMSCs+LV+AMP and ADMSCs+CD73siRNA+AMP group The content of adenosine(ADO)in the supernatant was detected by liquid chromatography mass spectrometry4.Pressure ulcer models were prepared from 8-week-old mice with magnetic disks for 36 h.They were randomly divided into model group(NC),ADMSCs transplantation group,CD73 activity inhibition group(ADMSCs+APCP),and empty virus control group(ADMSCs+LV)and CD73 expression inhibition group(ADMSCs+CD73 siRNA),5 in each group.Multipoint injection of 1×106 cells(100 μL)was used for treatment,and the NC group was given the same amount of PBS5.At 3d,7d,and 14d after treatment,the wound was photographed with a stereo microscope to calculate the wound healing rate;HE staining was used to observe the morphology,inflammatory cell infiltration,and angiogenesis;immunohistochemical measurements of pressure ulcers and surrounding CD31 and VEGF expression;Q-PCR was used.Tissue IL-10 and TNF-α expressions were detectedResults1.ADMSCs have a long spindle shape.Virus-transfected 72 h ADMSCs highly expressed GFP,and the CD73 gene and protein in the ADMSCs+CD73 siRNA group were lower than those in the ADMSCs+LV group,which was statistically different(P<0.001)2.Compared with ADMSCs+AMP group,the adenosine content in ADMSCs+APCP+AMP group is lower but higher than ADMSCs group,there is a statistical difference(P<0.001).Compared with ADMSCs+LV+AMP,ADMSCs+CD73siRNA+AMP.The adenosine content in the supernatant of the group was low and there was a statistical difference(P<0.001)3.At 3d,7d,and 14d after transplantation,the healing rate of ADMSCs group was higher than that of NC group and ADMSCs+APCP group,which was statistically different(P<0.05).Compared with ADMSCs+LV group,ADMSCs+CD73 siRNA was lower and there was statistical difference(P<0.05)4.At 3d,7d,and 14d after injury,the average area density of CD31 and VEGF immunohistochemical staining in the ADMSCs group was greater than that in the ADMSCs+APCP group,the lowest in the NC group,and there was a statistical difference between the groups(P<0.05);the area density of the ADMSCs+LV group was high There was a statistical difference in the ADMSCs+CD73 siRNA group(P<0.05)5.At 3d,7d,and 14d after injury,the relative expression of TNF-α mRNA in the ADMSCs group was lower than that in the ADMSCs+APCP group,the highest in the NC group,and there was a statistical difference between the groups(P<0.05);compared with the ADMSCs+LV group,ADMSCs The+CD73 siRNA group was high and there was a statistical difference(P<0.05)The relative expression of IL-10 mRNA in the ADMSCs group was higher than that in the ADMSCs+APCP group,and the NC group was the lowest.There was a statistical difference between the groups(P<0.05).Compared with the ADMSCs+LV group,it was lower than the ADMSCs+CD73 siRNA group,with a statistical difference(P<0.05)Conclusions1.Application of CD73+ADMSCs can promote wound healing,and inhibiting the expression or activity of CD73 on the surface of ADMSCs will slow healing2.CD73 promotes high expression of VEGF and CD31 in wounds,and accelerates local angiogenesis,which may be one of its mechanisms for treating refractory pressure ulcers3.CD73 promotes wounds with high expression of IL-10 and low expression of TNF-α.CD73+ADMSCs may regulate the immune microenvironment of wounds through the CD73/adenosine axis and promote healing of intractable pressure ulcers.