The Effects Of Nurr1 Gene On In Vitro Co-culture Of Neural Stem Cells And Microglia In Transwell

Objective:To study the interaction and effects of Nurrl-overexpressed neural stem cells and Nurr1-overexpressed in the co-culture of Transwell.Methods:1.Culture of the primary microglia cells:The cerebral cortex of SD rats on postnatal day 1-2 were isolated.The tissue is minced into a fine slurry using a pair of coneal scissors then followed by enzymatic disaggregation with 2 ml of trypsin/EDTA(0.05%/0.02%).DMEM with 10%FBS is added to the suspension(1:1 ratio)to neutralize the trypsin.The single cell suspension is obtained after this fluid is passed through a 70?m cell strainer.The cells are plated at the density of 5×106/mL in complete microglia cells medium.After 14 days mixed culture,microglia were separated by gentle shaking and immunoreactived for CD11b.2.Culture of the primary neural stem cells:The tissue of ventral mesencephalic is isolated from the E12.5-14.5 SD rats.Pipette tissue up and down several times with a sterile 1ml pipette after it is micro-dissected into a fine slurry using a pair of corneal scissors.The single cell suspension is obtained after this fluid is passed through a 70?m cell strainer.The cells are plated at the density of 2×105/mL in complete NSC medium supplemented with 20ng/mL epidermal growth factor(EGF)and 20 ng/mL basic fibroblast growth factor(bFGF).The primary neurospheres are harvested after 7 days and identified by immunofluorescence and for further differentiation culture.3.Nurrl primers is designed and amplified by PCR.Lentiviral vectors(pCDH-CMV-MCS-EF1-copGFP and pCDH-CMV-MCS-EF1-copRFP)and Nurrl gene is subsequently digested with Xba I and Not I,and then blunted with T4 DN A polymerase.The lentiviral vector pCDH-copRFP-Nurrl and pCDH-copGFP-Nurrl were established.Neural stem cells and microglia were transfected by these lentiviral vectors respectively.4.The Transwell co-culture system is constructed,and microglial cells are cultured in the upper chamber as neural stem cells are cultured in the lower chanber.These cells are randomly divided into six groups as following:mNSC group(control group),mNSC co-cultured with microglia(mNSC+MG group),mNSC co-cultured with Nurrl-overexpressed microglia(mNSC+NMG group),Nurrl-everexpressed mNSC group(NmNSC group),Nurrl-overexpressed mNSC co-cultured with microglia(NmNSC+MG group)and Nurrl-overexpressed mNSC co-cultured with Nurrl-overexpressed microglia(NmNSC+NMG group).mNSCs are collected after 3,6 and 9 days culture,and the expression of Otx2,Pitx3,TH and DAT was detected by Western blot,RT-PCR and immunofluorescence respectively.Results:1.The primary neural stem cells and microglia are established in vitro.Neural stem cells are immunostained with Nestin.These cells derived from 7 days differentiation culture are immunostained with Tuj1 and GFAP respectively.And microglial cells were immunostained with CD11b.2.Neural stem cells and microglial cells are overexpressed with Nurr1 respectively by lentiviral vectors.DA neurons increased after neural stem cells are overexpressed with Nurr1.The inflammatory factors decreased and neurotrophic factors increased after microglial cells are overexpressed with Nurr1.3.The expression levels of Otx2,Pitx3,TH,and DAT are significantly increased in Nurrl-overexpressed NSCs co-cultured with Nurrl-overexpressed microglia as it compared to the other groups,especially those goups do not transfected with Nurr1.These findings suggest that the DA neurons increased in Nurrl-overexpressed NSCs co-cultured with Nurrl-overexpressed microglia.Conclusion:Overexpression of Nurr1 in NSCs co-cultured microglia in vitro promotes NSCs differentiated into DA neurons,not only by reduce the levels of pro-inflammatory cytokines but also increase the level of neurotrophic factors.