Experiment Study On BFGF And RA To Induce The Differentiation Of Cord Blood Mononuclear Cells Into Neural Cell

Objective:1.To discuss the law and the feature of Separation,cultivation and amplification of the cord blood mononuclear cells (CB-MNCs)in vitro,and discuss the conditions of CB-MNCs differentiating into neural cells basically in vitro.2. Carry out the induced experiments in vitro of cord blood mononuclear cells (CB-MNCs) with all-trans-retinoic acid (RA) and basic fibroblast growth factor (bFGF) as an inducer. To discuss the mechanism of induced CB-MNCs differentiating into neural cells in vitro, which provide the experimental basis to the cell transplantation treatment of spinal cord injury.Methods:1.Collection of cord blood:Collecting cord blood of newborn who full-term pregnancy and normal delivery and passed the test according to the "blood medical standards" promulgated by the Ministry of Health, to collect umbilical cord blood completely closed after disinfection in the 3 stages of labor,use CPD21 anticoagulant and taken the average amount 80-130ml.2.Cord blood separation:Isolating fresh cord blood within 24 hours.Fresh blood mix with PBS by 1:1 ratio to produce a cell suspension, superimposed on Lymphocyte separation medium,2000 r/min centrifugation 20 min,then remove the white film, 1000 r/min centrifugation 10 min, discard supernatant, and then adjust the cell concentration to 1×106/ml.3 Group:Using Above-mentioned CB-MNCs with 3×106 cells inoculated in 6-hole plate, each hole final volume 3ml (containing 20% fetal bovine serum(FBS) in H-DMEM,Two antibiotic,penicillin and streptomycin each 100 U/ml);Divide into 4 groups according to the different combinations of induction factors in induction system. Group A:control group, without any factor. Group B: Add RA.Group C:add bFGF. Group D:add RA and bFGF. Observing the morphological changes of cells daily under the inverted microscope, and changed the medium every 3 days, discard the cells which not adherent. Observing the results after 2 weeks.4.Immunofluorescence identification:Detecting the expression of the neuronal markers Tuj-1 and the astrocyte marker GFAP, after induced the cells above-mentioned four groups 2 weeks later, using the method of immunofluorescence, observe by the Fluorescence microscope and take photographs.Checking 100 cells randomly in each group under the Fluorescence microscopy, and counting the amount of the cells Tuj-1-positive and GFAP-positive. Statistical analysis:Data are Expressed by x±s,and analysis single factor variance with SPSS 17.0 software.Results:1,The result observed under the microscope morphological: newly isolated CB-MNCs were small and round.1 day later, Small number of cells adhered to the wall with no significant morphological changes at the early culture;in the sixth day,the B,C,D groups were seen some cells out of synapses, and some were spindle,and some out of thin filamentous processes;in the tenth day, group D cells of some cells become larger cell body, some cells has elongation, some cells double poles, and some multi-polar; Fourteen days later, cells of A group have no significant changes,while B,C,D group showed a greater number of cell body, some has several long and big processes, even has the adjacent reticular cell processes, similar to neural cells.2 immunofluorescence results:CB-MNCs induced to neural cells, by way of statistical analysis, 1)The differentiation rate of D group after induced is the highest, and significantly different with other groups (P<0.05);(2) B,C,D group differentiation rate were significantly different with A group (P<0.05);(3) B and C groups showed no significant difference (P> 0.05).Conclusion:1.Both RA and bFGF could induce CB-MNCs to differentiate into neural cells.2.RA and bFGF can synergistically induced CB-MNCs to differentiate into neural cells,and the difference with the other groups is statistically significant.