Study On Factors Affecting Proliferation And Differentiation Of Neural Stem Cells In Vitro

Objective: To determine the effects of bFGF,EGF and N2 ,on the growth of neural stem cells.To study the regulation of bFGF,ATRA,VD3,DEX and BMP-2 on the differentiation of neural stem cells and the relation between the expression of retinoic acid receptor mRNA and the processes of differentiation.Methods: The effects of different neurotrophic factors on the growth and differentiation of neural stem cells were observed by cells counting and immunofluorescence staining.The levels of RARa mRNA and RXRa mRNA in differentiated neural stem cells were assayed by RT-PCR .agarose gel electrophoresis and image analysis. Results:1. Among the eight different groups containing bFGF,EGF or NI, group bFGF and group b*E*N were preferable. bFGF was essential for the growth of neural stem cells;EGF stimulated neural stem cells to differentiate and N2 induced abundant single cells,which neither formed neurospheres nor attached to the wall and differentiated,but degenerated and died at last.2. In the process of differentiating,if depriving of bFGF, neural stem cells died quickly;BMP-2,ATRA and R*V*D affected the differentiation of neural stem cells significantly;ATRA induced neural stem cells to differentiate into neuronic cells, VD3 and DEX also induced neural stem cells to differentiate into neuronic cells,but not as effective as ATRA;Neural stem cells treated by BMP-2 differentiated into astrocytes partially; ATRA and BMP-2 antagonized each other in their effects on neural stem cells differentiation.3. The bFGF could not enhance the expression of RARa mRNA,but could affect the process of expression of RARa mRNA induced by ATRA or BMP-2; RARa mRNA increased significantly in neural stem cells induced by either DEX or R*V*D;Even thought BMP-2,ATRA and VD3 could also affect the expression of RARa mRNA or RXRa mRNA, there was no statistic meaning.4. The numbers of differentiated cells induced by BMP-2,ATRA,VD3 and DEX were not correlative with the amount of RA receptor mRNA.Conclusion:1. bFGF is essential for the proliferation and differentiation of neural stem cells, it can also affect the process of expression of RARa mRNA induced by ATRA or BMP-2;2. Neural stem cells can be induced to differentiate into neuronic cells by ATRA,VD3 and DEX,and into astrocytes by BMP-2 ; ATRA and BMP-2 antagonize each other.3. DEX and R* V*D enhance the expression of RARa mRNA significantly.4. The differentiation effects of neural stem cells treated by BMP-2, ATRA,VD3 and DEX are not correlative with the expression of RA receptor mRNA.