The Experimental Study On Inducing Rat BMSCs To Differentiate Into NSCs By AFGF

PART ONETHE CULTURE, PURIFICATION AND IDENTIFICATIONOF RAT BMSCs IN VITROObjective: To culture in vitro and purify rat bone marrow mesenchy-mal (BMSCs), study their biological characteristics, and detect the cellsurface markers to identify the cells.Methods: Rat BMSCs were cultured by the whole bone marrowadherent culture method, and were purified through subculture. The cellmorphological characteristics were observed under the inverted phasecontrast microscope; The cell growth curves were drawn by microeulturetetrazolium (MTT) assay; The cell cycle of the3rd generation cells andsurface markers were detected with flow cytometry (FCM).Results: The cultured cells showed adherent growth and long spindleappearance.The growth curve of the1st,3rd,5th-generation cells showedS-shape and no significant difference in activity. FCM showed that therewere94.34%of the cells in G0/G1phase of the cell cycle, having strong proliferation ability. FCM detected that CD29, CD54and CD90werepositively expressed, but CD45was negatively expressed.Conclusion: The whole bone marrow adherent culture method cancultivate highly purified rat BMSCs with stem cells’ characteristics, whichcan grow stably, proliferate rapidly, and be multiple-passaged. PART TWOINDUCING RAT BMSCs TO DIFFERENTIATE INTONSCs BY aFGF AND IDENTIFING THE NSCsObjective: To study the feasibility, the best effect dose and the optimalinduction time that acidic fibroblast growth factor (aFGF) induced rat bonemarrow mesenchymal stem cells (BMSCs) to differentiate into neural stemcells (NSCs), and identify the cells before and after differentiation.Methods: Rat BMSCs were cultured by the whole bone marrowadherent culture method. The experimental group was the aFGF inducedgroup-A group:10,20,40,60,80,100,120ng/ml aFGF were used forinduced differentiation. The positive control group was the bFGF inducedgroup-B group: with20ng/ml bFGF-induced differentiation. The negative control group-N group hadn`t inducers. The morphological changes of thecells before and after induced were observed under the inverted phasecontrast microscope. Reverse transcription-polymerase chain reaction(RT-PCR) was used to detect the gene expression of Nestin andneuron-specific enolase (NSE). The protein expression of Nestin and NSEwere detected by the immunofluorescence staining method.Results: RT-PCR results showed that Nestin mRNA and NSE mRNAwere expressed in different groups, and the aFGF`s best effect dose was80ng/ml. With the best effect dose completed the subsequent induceddifferentiation experiments. Compared with other groups, RT-PCR resultsshowed that6hours after induction the Nestin mRNA expression of Agroup were highest(P<0.05), the difference was statistically significant.With the extension of the induction time,Nestin mRNA expression wasgradually weakening trend. After induced6d, the NSE mRNA expressionof A group compared with other groups were highest(P<0.05), thedifference was statistically significant. After induced6h visible minoritycell bodies had contracted to become oval and extend elongated protrusions.Immunofluorescence showed a positive expressed of Nestin. Induced cellsafter6d showed bipolar or multipolar, intercellular ridges were connectedinto a net, immunofluorescence staining showed the protein expressed ofNSE was positive. Conclusion: aFGF can culture the rat bone marrow-derived NSCs invitro, is an effective biological inducers, the best dose of aFGF is80ng/ml.The differentiation cells can continue to differentiate into neuron-like cells,which identified NSCs`differentiation function. Rat BMSCs induced byaFGF after6h with NSCs characteristics can prove that the aFGF optimalinduction time is6h.