Differentiation Of Rat Marrow Mesenchymal Stem Cells Into Neural-like Cells In Vitro

Objective To research MT and supernatant of OECs introduce marrow mesenchymal stem cells into neuron-like cells after multiplication and chondrogenesis.Investigate MT introduce marrow mesenchymal stem cells into neuron-like cells on different time And to explore MT and supernatant of OECs introduce marrow mesenchymal stem cells into neuron-like cells.Find out the best program of introduction.The experiment provided some foundat ion about introducing marrow mesenchymal stem cells into neuron-like cells.And the experiment aslo provided another seed-cells for MSCs transplantation.Methods We obtained bone marrow from healthy male adult SD rats in bacteria-free environment.Then we used the 1.077g/ml Ficoll to get the nucleated cell layer by density gradient centrifugation.The cells were Inoculated in culture bottle with 10%FBS L-DMEM culture medium at the seeding density of 5×10⁶/cm² and the bottles were placed in the 37℃incubator with 5%CO₂.After 48 hours' culture,we changed the half mount medium and abandoned the inadherent cells.The medium was changed every 2 or 3 days and the cells would be subcultured at 1:3 rate when they reached 90%fusion growth.(1) We introduced the passages 2 MSCs into neuron-like cells with the 10 mmol/L MT when they reached 90%fusion growth.We observed the cells' sharp with invert microscope after introduced 3, 7,10,14 days.The expression of nestin,NF and GFAP in the differentiated cells was also detected by immunof]uorescence after introduced 3,7,10,14 days.(2) We introduced the passages 2 MSCs into neuron-like cells with the 10 mmol/L MT when they reached 90%fusion growth.We observed the cells' sharp with invert microscope.The expression of nestin,NF and GFAP in the differentiated cells was also detected by immunofluorescence after introduced 14 days.And westernblot as used to detecte neuron-like cells which have been introduced.At last, we analyzed the differences among the groups statistically.Results The MSCs were spindle shaped,also some were circular, multipolarity,a little had some short and small towers.(1) Three days after induction with MT some cells were found to contract,refract more light and protrude and bulge.Some neuron-like cells contracted to atriangular shape,and some others became simple bipolar or multipolar cells.Some long towers' terminal growed some tubercles like cone,and appeared neuron-like sharpe after 7 days.Moer and more neuron-like cells were introduced,towers longed after 10 days.Neuron-like cells increased,some cells' towers connected like reticular.After being induced for 3 days,the cells expressed nestin.After 7-14 days,the cells expressed NSE and NF,but didn't express GFAP..(2) After introduction with MT and supernatant of OECs 3 days,MSCs differentiated into neuron-like cells gradually,and appeared neuron-like sharpe.Some neuron-like cells can express NF and NSE,but didn't express GFAP.The MT and supernatant of OECs group changed remarkably,the wasternblot detection can proved also.Conclusion(1) MT can introduce MSCs into neuron-like cells.Process: MSCs→neuron-like stem cells→neuron-like cells.(2)MT and supernatant of OECs can introduce marrow mesenchymal stem cells into neuron-like cells.The MT and supernatant of OECs group is better than MT group,supernatant of OECs group.