Regulatory Effects Of P2X7 Receptor On Adipose Derived Stem Cells Differentiation To Schwann Cells

Objectives:Adipose-derived stem cells(ADSCs)are ideal cells for nerve transplantation to treat nerve defects,but the high mortality rate after transplantation is an urgent problem.The P2X7 receptor has been a hot target for neurological disease research.P2X7 receptors have been known to save ADSCs from the ATP-induced death mediate the differentiation of Schwann cells into myelin sheath,and promote myelin sheath formation.The purpose of this experiment is to study whether P2X7receptor can promote the differentiation of fat stem cells into Schwann cells,enhance the effect of fat stem cells on the function of promoting nerve regeneration after differentiation,and provide a theoretical basis for the search of drug intervention targets for nerve repair cell therapy.Methods:Subcutaneous adipose tissue of the abdomen of male Sprague-Dawley(SD)rats was digested with type I collagenase,cultured and passaged.ADSCs at the third passage were used in subsequent experiments.The third generation of undifferentiated adipose-derived stem cells(uADSCs)was transfected with P2X7R lentivirus.The uADSCs model with high expression of P2X7R was constructed,and Western blot was used to verify if the transfection of the P2X7R-uADSCs was successful.Proliferative capacity of infected and uninfected ADSCs was evaluated by MTT.uADSCs and P2X7R-uADSCs were transferred to a six-well plate at a density of 1×10~5/mL,treated with 1 mM?-mercaptoethanol for 24 h,35 ng/mL all-trans-retinoic acid was added.After cultivation for 72 h,followed by a fresh DMEM/F12 medium contained 5 ng/mL basic fibroblast growth factor,10 ng/mL platelet-derived growth factor,14?M forskolin,and 200 ng/mL heregulin.The cells were maintainted for 1-2 weeks.RT-PCR was used to detect the mRNA levels of neurotrophic factors BDNF,NRG-1 and NT-3,neurotrophic factor receptor TrkB and TrkC,myelin related proteins P0 and PMP22 in dADSCs and P2X7R-dADSCs.Western blot analysis was performed to determine the expression of neurotrophic factors NT-3 and NRG-1,neurotrophic factor receptor TrkC and ErbB-4,myelin related proteins P0 and MAG in dADSCs and P2X7R-dADSCs respectively.Results:Primary ADSCs were extracted by type?collagenase digestion,and cultured in DMEM/F12(containing 10%fetal bovine serum FBS,1%penicillin-streptomycin mixed solution)culture medium,the cell attachment speed was quick.Over about 4 to 5 hours of incubation,a small quantity of dispersed cells can be seen adhered to the wall.After 48 hours,the morphology of ADSCs cells was observed under an inverted microscope,and they were basically adherented.Most of the shapes were short spindle,star-shaped and irregular polygonal structures.After 5to 7 days,ADSCs were spindle-shaped,growing vigorously,and the cells grew rapidly after passage.uADSCs were successfully transfected with P2X7R lentivirus in vitro,and continuous fluorescence was observed by fluorescent microscopy.The testing results through Western Blot revealed that the expression of P2X7 protein in the uADSCs group and the GFP-uADSCs group was significantly lower than that in the P2X7R transfection group.There was no significant difference in cell proliferation between P2X7R-uADSCs and uADSCs by MTT.uADSCs and P2X7R-uADSCs were successfully induced in specific medium.Differentiated into Schwann-like cells,the morphology of cells was long and spindle-shaped,and the cells grew spirally.RT-PCR showed that neurotrophic factors BDNF,NRG-1 and NT-3,neurotrophic factor receptor TrkB and TrkC,and myelin proteins P0 and PMP22 were expressed in dADSCs and P2X7R-dADSCs.The mRNA expressing level of factors in P2X7R-dADSCs group was significantly higher than those in the dADSCs group(P<0.05).Western blot showed that the protein expressions of neurotrophic factors NT-3and NRG-1,neurotrophic factor receptor TrkC and ErbB-4,myelin protein P0 and MAG were increased in P2X7R-dADSCs group compared with dADSCs group(P<0.05).Conclusion:P2X7R has no significant effect on the proliferation of ADSCs,but it can enhance the expressions of neurotrophic factors and their corresponding receptors in ADSCs and promote myelin formation after differentiation,which is conducive to enhancing the capability of nerve repair of ADSCs.