Experiment Study On β-ME And BFGF To Induce The Differentiation Of Bone Marrow Mesenchymal Stem Cell Into Neurons Cell In Vitro

Object:1.The experimental studies the characteristics of the bone marrow mesenchymal stem cells isolation,cultivation and expansion in vitro,The establishment of SD rat bone marrow mesenchymal stem cells cultured in vitro system.2.Respectively use of beta-Mercaptoethanol (β-ME) and basic fibroblast growth factor(bFGF) as an induction agent,Differentiate the bone marrow stem cells in to neurons and astrocytes in vitro successlly.Discuss the mechanism ofβ-ME and bFGF to induce thedifferentiation of bone marrow mesenchymal stem cell into neurons cell,provide experimental basis for spinal cord injury of cell transplantation treatment.Method:1.Isolation,purification and expansion of the rat BMSC.1.1 Primary culturePrimary culture of rat BMSC were collected from the femurs and tibias of one month old SD rat.The bone marrow was washed from marrow cavity with L-DMEM complete medium.After filtrated,the marrow suspension were collected and placed in a 25 cm2 flask.The culture were maintained at 370C 5%C02 and saturated humidity.The non-adherent cells were removed by replacing the medium after 72 hours of culture and thereafter to replace the medium every 72 hour.When the cells had grown to confluence(about 14 days),they were passaged by 0.25%trysin and replated after dilution to 1:2 or 1:3.The passaged rBMSC atached to plastic flask within 12 hours and reached confluence within 7 to 10 days.1.2 Capability of proliferation of rat BMSCThe 1th,4th,7th,10th and 13th generation BMSC were selected to be investigated their capability of proliferation curve and adherence rate. The result showed1.3 The antigenic phenotypes of rat BMSCThe 4th generation cells were collected and their expressions CD90 and CD44 are positive,CD34 and CD45 are negative.2.The differentiation of rBMSC in vitro2.1 Differentiate rBMSC into the neurons cellsThe 4th generation rBMSC cells were collected,by2×104/ ml concentration were inoculated in 24-well plates until the cells reached 70%～80%fusion,addingβ-S-ethanol(β-ME) and basic fibroblast growth factor(bFGF) respectively,inverted phase contrast microscope for observation the changes of the cell morphology.2.2 Immunofluorescence identification of cellsusing cells immunofluorescence method,after the 5h cells of these two groups to further test markers TuJ1 neurons and astrocytes marker GFAP expression,observe them under fluorescence microscope and photographed record. 2.3 the positive rate of rBMSC cells differentiate into neuronsFluorescence microscope check 300 cells in a random,count the number of TuJ1-positive cells,testing the group differences at different time points,and each time point the differences between the groups. Statistical analysis of data that are x±s,by software SPSS 13.0.2.4 Immunoblotting(Western blotting) detection of the differentiation cell specific markersRespectively,after differentiation of the cells 0h,1h,5h,using of 2X SDS sample buffer cracker cells for neuron-specific protein TuJ-1 and astrocytes-specific quantitative analysis of GFAP protein.Result:1.adherent screening method,through replacement of the liquid can be repeated for separation,purification of BMSC,the method is simple,convenient and effective.2.BMSC have a strong proliferation ability in vitro,former than The 10th generation have the same cell morphology and proliferation ability,with the increase in the number of passages,the ability of poliferatrion weakened and irregular changes in morphogenesis.Cell growth adherent of the generation of 1,4,7,10,13 is basically the same as the rate of change,no significant difference.3.The antigen characteristic of rBMSC surface markers present diversity, rBMSC hematopoietic cells did not express surface antigens CD34,CD45, show that rBMSC is non-hematopoietic cells;while express surface markers of CD90,CD44,show that the characteristics of rBMSC is non-uniformity.It expresses the surface markers of the mesenchymal cells,endothelial cells and epidermal cell.4.adding inducer into 4th generation rBMSC,after cells differentiate into neurons and astrocytes. Observed under visible light:the former cells smaller,often have a few short processes and a longer process,showing the typical bipolar, multipolar and pyramidal-shaped structure,strongger refractive index; latter cells larger,have cell processes and more rough,and some were stellate shape,flat on the dish.5.cells immunofluorescence showed that after 5h differentiation,some TuJ1-positive cells,were neurons;some GFAP-positive cells,were astrocytes.6.rBMSC cells induced into neurons positive rate,by the statistical analysis,β-ME group after 1h,5h the highest rate of groups,24h bFGF group the highest rate of groups, were significantly different(P<0.01);β-ME group at 24h after induction of the majority of cells had died,not statistics.β-ME group 1h and 5h of the differentiation rate were significantly different(P<0.01);bFGF group 5h and 24h of differentiation rate were significantly different compared 1h(P<0.01),but 5h and 24h,the division was no significant difference (P>0.05).7.Western Blotting test results show that in the 20ug total protein samples,with the induction time,rBMSC induced differentiation, the expression of TUJ-1 and of GFAP marked were increased in the amount.And,with the time-induced stem cells to differentiate into neurons and astrocytes increase in the number. Conclusion:1.The experiments using bone marrow culture method for direct isolation and culture of BMSC,this method is simple,the effect was reliable,and through the passage of several generations will be able to obtain amplification of high purity,high number of rBMSC,meet the needs of tissue engineering experimental research.2.The use of beta -Mercaptoethanol(β-ME) and basic fibroblast growth factor(bFGF) as an induction agent,can induce Bone mesenchymal stem cell(BMSC) differentiate into neurons and astrocytes in vitro.