The Effects Of BDNF In The Proliferation And Differentiation Of NSCs

Neural stem cells(NSCs) were defined as undifferentiated neural cells that were endowed with a high potential of proliferation and the capacity for self-renewal,they also could differentiate into the main phenotypes of the nervous system:neuronal,astrocytic and oligodendrocytie.It has vast clinical application foreground for it's ability of renew and differentiation. Recent studies had shown that transplantation of NSCs into the injured spinal cord could promote functional recovery in adult animal models.However,the majority of transplanted NSCs differentiated into astrocytes,which resulted in the formation of glial scar in the injuried spinal cord and prevented the recovery of neurofunction. BDNF(brain - derived neurotrophic factor )is a low basic protein which was initially extracted from swine brain in 1982.It is one of the most representative member of neurotrophic factor family.Many researchs has shown that BNDF can promote the differentiation of neural stem cells into neurons.For above reason, this study tried to pay more attention to NSCs and BDNF. The aims of this study were: to investigat the roles of BDNF in the proliferation and differentiation of NSCs. Simultaneously,we discuss the role of TrkB and p75, receptor of BDNF,in the differentiation of NSCs.There are two parts in this articles: Part 1 The isolation, cultivation and identification of neural stem cells in vitroObjectiveTo master the method of NSCs culture in vitro. observed the character of NSCs growth and the structure under light microscope and electron microscope.Methods1 NSCs were isolated from the brain of postnatal SD rats within 3 days.2 Observed the effect of different cultured density and passage methods on neural stem cells in vitro.3 Identification of neural stem cells using immunocyto- -chemistry of Nestin,Brdu, NSE and GFAP.4 Observed the ultrastructure of undifferentiated and differentiated NSCs under transmission electron microscope.Results and Conclusion1 Most of cultured cells were Nestin-postive, had capacity of self-duplication and could differentiate into the main phenotypes of the nevous system. The introduced method is simple,operative and reliable .2 The optimal cultured density was 1×106 /ml, the method of passage should depend on your purpose. The time was about 7 days.3 Observation with TEM: Before differentiation, the nucleus/cytoplasm ratio was very high, The nucleus was ellipse or round. Cells had very little cytoplasm and organelle but with more ribosome.After differentiation, the volume of cytoplasm increased dramatically and various kinds of organelles appeared in the cytoplasm such as Golgi complex, microfilament, microtubule.Part 2 The effect of brain - derived neurotrophic factor on the proliferation and differentiation of neural stem cellsObjectiveour researches was focused on the effect of BDNF on the proliferation and difference of neural stem cells. The optimal concentration and time of BDNF should be find in the differentiation of NSCs. Simultaneously,we discuss the receptor of BDNF: TrkB and p75 in the differentiation of NSCs.Methods1 The effect of different concentration of BDNF on the proliferation of NSCs at different time point was studied with MTT assay.draw the growth curve of neural stem cells. The cultural cells was divided into serveral groups that inclued 0,2,20,200ng/ml groups according to the doses of brain- derived- neurotrophic factor.within this groups ,0ng/ml was control group.2,20,200ng/ml groups were experimental groups.2 Using immunocytochemistry to study the effect of different concentration of BDNF on differentiation the of NSCs at different time point. The groups was same as the MTT assay described above. The differentiated cells were harvested at 1d,3d,5d,7d after induction. Immunocytochemistry was carried out to analyze the expression of NSE in those cells. Meanwhile, the percentage of NSE positive cells was calculated.3 The expression of trkB,P75 mRNA was investigated by RT-PCR. The cells was divided into three groups: undifferentiated NSCs, differentiated NSCs with no inductor. differentiated NSCs with 20ng/ml BDNF.all differentiated NSCs were detected at the time of 1d,3d,5d after induction. undifferentiated NSCs were detected at 3d after passage.Results1 MTT assay: Compared with control group, every experimental groups have no significant difference in each time point.2 immunocytochemistry:With the increasing concentration of BDNF ,the percentage of NSE positive cells was also increased. Statistical analysis showed a significant difference between each groups.we aslo found that the proportion of NSE-positive neuron in each group reached its peak on the 3rd day,which was approximately 80%,but only about 35% was found in the control group.3 The results of RT-PCR: In the undifferentiated NSCs,both trkB and p75 were expressed. in the differentiated NSCs, compared with no inductor the expression of trkB was significantly increased in the group adding 20ng/ml BDNF. but p75 did not show up this phenomenon.Conclusions 1 BDNF has no effect on the proliferation of neural stem cells.2 BDNF can noticeable contribut to the differentiation of neural stem cells.the proportion of differentiation depended on the concentration of BDNF and reached its peak on the 3rd day.3 The Promoting effect of BDNF on the differentiation of NSCs into neurons maybe related to the increasing expression of trkB mRNA.in this process, p75 have no apparentely role.