The Effects Of BDNF-overexpressing Neural Stem Cells Transplantation On BDNF Downstream Receptors Expression And Naptogenesis In The Hippocampus Of Exposed Rats

Part I.Isolation,culture,differentiation and identification of neural stem cells in vitroBackground and objective:The inhibition of neurogenesis caused by radiation is considered to be the most importment in the mechanisms of radiation-induced cognitive dysfunction.Neural stem cells(NSCs)transplantation is expected to be the most effective method to improve radiation-induced cognitive dysfunction.Neural stem cells are a kind of primordial cells with the potential ability to self-renew and differentiate into neurons,astrocytes and oligodendrocytes.Therefore,The key is to isolate and culture the cells in vitro.Methods:Our research group had isolated neural stem cells from the cerebral cortex of Sprague Dawley fetal rats aged 14 to 15 days.Apart from amplification in vitro,the cells were differentiated.Then cultured neural stem cells were identified using Nestin immunofluorescence staining technique.Neurons after differentiation were identified using marker Neuronal Class ? ?-Tubulin(Tujl)and astrocytes after differentiation were identified using marker glial fibers acidic protein(GFAP)by immunofluorescence staining technique.Results:Nerve balls grew in suspension were positive for Nestin immunofluorescence staining.Under serum-containing medium,they were differentiated,with nerve cell morphology,and they were positive for Tuj 1 and GFAP immunofluorescence staining.Conclusion:Neural stem cells isolated and cultured from fetal rats had the abilities of self-renewal,proliferation and differentiation in vitro,which made preparation for the next experiment.Part ?.The effects of BDNF-overexpressing neural stem cells transplantation on BDNF downstream receptors expression in the hippocampus of exposed ratsBackground and objective:Brain-derived neurotrophic factor(BDNF)plays a neurotrophic role by combining the downstream receptors mainly.Our research group had found that the expression of BDNF increased after BDNF-overexpressing neural stem cells were transplanted into hippocampus of exposed rats.In this experiment,the expressions of downstream receptors TrkB and P75NTR of BDNF in hippocampus were detected by PCR and Western blot at 2,8 and 14 weeks after transplantation.Methods:The rats received a single dose of 20Gy whole brain irradiation.Forty one-month SD rats were randomized into four groups:control group(C group),irradiated group(R group),GFP-modified NSCs transplantation group after irradiation(R+GFP-NSCs group),and GFP-BDNF modified NSCs transplantation group after irradiation(R+GFP-BDNF-NSCs group).Neural stem cells were transplantated after 4 weeks irradiation.The expressions of BDNF,TrkB and P75NTR in hippocampus were detected by PCR and Western Blot at 2,8 and 14 weeks after transplantation.Results:Compared with the irradiation group,the expression of BDNF in the hippocampus of the R+GFP-BDNF-NSCs group was significantly higher than that of R group at 2,8 and 14 weeks after transplantation.The level of TrkB in the hippocampus of the R+GFP-BDNF-NSCs group was significantly higher than that of R group at 2,8 and 14 weeks after transplantation.At 2 weeks after transplantation,the expression of P75NTR was decreased,and no significant changes were observed at other time points.Conclusion:BDNF-overexpressing neural stem cells transplantation into the h ippocampus of exposed rats can increase the expression level of TrkB in hippocampus,but have little effect on the expression of P75NTR.Part ?.The effects of BDNF-overexpressing neural stem cells transplantation on synaptogenesis in the hippocampus of exposed ratsBackground and objective:BDNF can adjust the synaptic number,synaptic function and dendritic morphology through the mechanisms of presynaptic and postsynaptic function Synaptogenesis is the basis of the neural circuits.The neural circuits are the basis for the improvement of radiation-induced cognitive dysfunction.In this part of the research,we investigated whether the BDNF-overexpressing neural stem cells transplantation into the hippocampus of exposed rats could promote synaptic growth.Methods:The rats were given a single dose of 20Gy irradiation of the whole brain.Forty one-month SD rats were randomized into four groups:control group(C group),irradiated group(R group),GFP-modified NSCs transplantation group after irradiation(R+GFP-NSCs group),and GFP-BDNF modified NSCs transplantation group after irradiation(R+GFP-BDNF-NSCs group).Neural stem cells were transplantated after 4 weeks irradiation.The expressions of synaptophysin and PSD95 in hippocampus were detected by PCR and Western Blot at 2,8 and 14 weeks after transplantation.The expression of synaptophysin in DG area was observed by immunofluorescence at 8 and 14 weeks.Results:At 2,8 and 14 weeks after transplantation,the mRNA and protein expressions of synaptophysin in the hippocampus of the R+GFP-BDNF-NSCs group were significantly higher than that of R group.At 14 weeks after transplantation,the expression of PSD95 in the hippocampus of the R+GFP-BDNF-NSCs group was significantly higher than that of R group.At 8 and 14 weeks after transplantation,compared with the R group,the expression of synaptophysin in DG of hippocampus was significantly higher.Conclusion:BDNF-overexpressing neural stem cells transplantation into the hippocampus of rats exposed,could promote the expressions of synaptophysin and PSD95 at 14 weeks after transplantation,to some extent,could promote the synaptic growth in hippocampus.