Study On The Synaptic Plasticity Of Differentiate The Human Umbilical Cord Mesenchymal Stem Cells Into Neuron-Like Cells By Resveratrol In Vitro Induction

Objective : Extracting and purifying the human umbilical cord mesenchymal stem cells(hUC-MSCs)from umbilical cord tissue and culturing them in the laboratory to observe and record the morphological changes and growth situation;The use of flow cytometry for phenotypic analysis;To explore the effect and efficiency of different concentrations of resveratrol on the induction of differentiating human umbilical cord mesenchymal stem cells into neuron cells;To observe the morphological changes of neuron-like cells after induction,explore the synaptic plasticity of neuron-like cells after induction,and detect the expression level of genes and proteins in synaptic markers;Finally,to provide theoretical support for the transplantation of hUC-MSCs in clinical treatment of central nervous system injury and other diseases.Methods:Primary hUC-MSCs were obtained from umbilical cord tissue by enzyme digestion.h UC-MSCs phenotype was detected by flow cytometry.P3-generation hUC-MSCs were randomly divided into Groups A,B,C and D.Group A,B,C and D were added with pre-inducing solution(Neurobasal medium containing 50 ng/mL bFGF)for 24 hours,then add the concentration of resveratrol induction solution in to A,B and C groups for one day(the concentration of resveratrol of Group A is 5 mg/L;Group B is 10 mg/L;Group C is 20 mg/L).Then,the maintenance solution was changed and cultured for 7 days.The inducer was prepared with Neurobasal medium.The maintenance solution was prepared with Neurobasal medium containing 2% B27 and 50ng/mL bFGF.Group D use Neurobasal medium alone for 1-day induction,and then culture it for7 days with a maintenance solution(Neurobasal medium containing 2%B27 and 50 ng/mL bFGF).Use the inverted phase contrast microscope to dynamically observe the morphological changes of cells in each Group.Select ten non-overlapped visual fields randomly from each Group on the1,2,3,4,5,6,and 7 days after induction,to collect the images and count the neuron-like cells in the visual field;Detect the positive expression rate of Synaptophysin(SYN),growth-associated protein 43(GAP43),and postsynaptic density 95(PSD95)in each Group of cells by Immunofluorescence staining,then select a Group with better induction effect,extract total cellular RNA and protein on the 0th,1st,2nd,3rd,4th,5th,and 6th days of induction,and detect the expression of the situation of SYN,GAP43,PSD95 by RT-PCR and Western Blot methods.Results:1.Flow cytometry analysis results of P2 and P8 generation cell phenotypic: expression on CD105,CD90,and CD73;no expression on CD11 b,CD19,CD34,CD45,and HLA-DR(MHC-II).2.Detection of positive rates of SYN,GAP43 and PSD95 by immunofluorescence,The positive rates of Groups A,B,C,and D of SYN are 0%,3.24±1.40%,and 49.32±2.85%,0% respectively;The positive rates of GAP43 are 0%,3.16±1.42%,5.62±2.41%,0%respectively;The positive rates of PSD95 are 0%,2.88±1.60%,41.75±2.08%,0% respectively.The results showed that the positive rates of the three synaptic markers were the highest in the C group,and there was significant difference compared with other groups(P < 0.01).3.RT-PCR detected the relative transcription levels of SYN,GAP43 and PSD95 mRNAs in group C on the 0,1,2,3,4,5,and 6 days after induction,the relative transcription levels of SYN are 0.386±0.026,0.446±0.011,0.462±0.014,0.515±0.016,0.523±0.029,0.558±0.028,and0.516±0.034,respectively;the relative transcription levels of PSD95 are0.055±0.013,0.170±0.017,0.265±0.018,0.290±0.016,0.327±0.023,0.291±0.020,0.269±0.020;the relative transcription levels of GAP43 are0.082±0.018,0.112±0.014,0.119±0.012,0.124±0.017,0.399±0.020,0.559±0.017,0.387±0.019.the results showed that there was a significant difference between any time group after induction and the blank group(P<0.01).The three markers are all the same.4.Western Blot detected the relative protein expression levels of SYN,GAP43,and PSD95 in group C cells on the 0,1,2,3,4,5,and 6days after induction.The relative protein expression levels of SYN are0.185±0.017,0.346±0.027,0.535±0.035,0.598±0.026,0.678±0.037,0.657±0.031,0.585±0.034;The relative protein expression levels of GAP43 are 0.188±0.015,0.314±0.038,0.386±0.032,0.582±0.035,0.679±0.037,0.458±0.043,0.368±0.029 respectively;The relative protein expression levels of PSD95 are 0.065±0.020,0.303±0.036,0.397±0.032,0.497±0.041,0.649±0.053,0.489±0.060,0.263±0.025,respectively.After induction,each time group had significant difference compared with blank group(P<0.01).The three markers are all the same.Conclusions:1.The hUC-MSCs can be successfully isolated and purified from the umbilical cord stroma and can be stably grown and passage in a laboratory environment.2.Flow cytometry analysis results of h UC-MSCs: the surface markers of hematopoietic stem cell CD11 b,CD19,CD34,CD45,and HLA-DR(MHC-?)are expressed in negative;the surface markers of mesenchyme stem cell CD105,CD90,CD73 are expressed in positive.3.20mg/L resveratrol can effectively induce h UC-MSCs into neuron-like cells in vitro and form a synapse-like structure,synaptic markers(SYN,GAP43,PSD95)can be expressed.4.hUC-MSCs have a substance basis for synaptogenesis after induction,providing theoretical support for further cell electrophysiological experiments.5.hUC-MSCs have the ability to differentiate into neuron cells and can be used as a cell source for the treatment of stem cells transplantationfor neurological diseases.