Research On Differentiation Of Neural Stem Cells Into Dopaminergic Neurons By Microglia Of Nurr1 Gene Overexpression

ObjectiveTo investigate the influence of Nurrl gene on the activation state of microglia and overexpressing Nurrl gene of microglia for neural stem cells differentiate into dopaminergic neurons. To explore the possible mechanism of action. To acquire new ideas and methods for neural stem cells transplantation for Parkinson’s disease treatment, provide new ideas and ways to make treatment improved.Methods1. Dissecting the newborn (0-2d) SD rat cerebral cortex by trypsin digestion and mechanical pipetting into single cell suspensions, and were inoculated in medium containing 10% fetal bovine serum,7～10 days later purified microglia by mechanically shaking and differential adherence methods, and the cells were identified with immunocytochemistery for CD11b/c.2. Nurrl gene coding sequence was synthesized and cloned into the eukaryotic expression vector pCDH-MCS-T2A-copGFP-MSCV to establish recombinant plasmid pCDH-Nurrl. The recombinant plasmid was identified by restriction enzyme digestion and sequence analysis. Microglia were transfected with the recombinant plasmid by liposomal transfection regent.3. The microglia overexpressing Nurrl gene and the simple microglia were added LPS to activates, in the same time the culture medium was collected among the groups. ELISA analysis was carried out to detect the influence of Nurrl gene on secretion of inflammation-related factors(IL-1 and TNFa) and neurotrophic factors(BDNF, GDNF and PDNF) in microglial of different states.4. The ventral mesencephalon was dissected from embryonic day14(E14) rat embryo. The brain tissue was triturated into a fine single-cell suspension by mechanical separation method. The cells were cultured with the serum-free medium. The neural spheres of the third passage were identified with immunocytochemistery for Nestin. At the same time, the cells were inducted to differentiation, and the phenol types of differentiated cells were identified by immunocytochemistery for β-3-tubulin, GFAP and TH respectively.5. Using transwell system, the underlying cultured microglia, the upper cultured NSCs, and are grouped as follows:(1) NSCs and simple microglia co-culture; (2) NSCs and microglia overexpressing Nurrl gene co-culture. After cultured 3d,6d, 9d, the NSCs of two groups were observed for survival, growth, and differentiation to the DA neurons.6.These time points, to detect two groups NSCs differentiate into DA neurons by immunocytochemistry; detect the level of two groups NSCs differentiate into DA neurons related proteins(DAT、TH、FOXA2、Otx2、Pitx3、VMAT2、Lmxla) expression by Western Blot; and detect two groups NSCs differentiate into DA neurons maturation-related genes(DAT、TH、FOXA2、Otx2、Pitx3、VMAT2、 Lmxla) expression by real-time PCR.Results1. The mixed glial cells growth stratified after cultured to 7～10d, use mechanical shaking and differential adherence method to obtain purified microglial cells, immunocytochemistery for CD11b/c of positive, and the purity is 95%.2. Successfully constructed eukaryotic expression vector pCDH-Nurrl, by restriction enzyme digestion and sequence analysis, judged to be positive clones.3. ELISA analysis results show that the microglia of Nurrl gene overexpression can significant reduce inflammatory factors(TNF-a, IL-1) secretion than the simple microglial in that LPS-induced and can to increase neurotrophic factors(BDNF, GDNF and PDNF) secretion.4.7 days after primary culture, the cells derived from E14 rat embryonic mesencephalon form neurospheres which can be sub-cultured. A great many of neurospheres can obtained by suceessive passage. Immunocytochemistery showed the neurospheres were nestin positive, and after differentiation the cells expressed β-3-tubulin, GFAP and TH.5. After co-culture 3d,6d,9d, two groups NSCs were identified with immunocytochemistery method showed that the group of microglia overexpressing Nurrl gene and NSCs co-culture have more NSCs than the simpe microglia and NSCs co-cultured group can differentiate into DA neurons.6. These time points, Western blot showed that the NSCs differentiate into DA neurons related proteins(DAT、TH、FOXA2、Otx2、Pitx3、VMAT2、Lmx1a) expression level of the group of microglia overexpressing Nurrl gene and NSCs co-culture were better than the simple microglia and NSCs co-cultured group.7. These time points, Real-time PCR results showed that the NSCs differentiate into DA neurons maturation-related genes(DAT、TH、FOXA2、Otx2、Pitx3、VMAT2、 Lmxla) expression of the group of microglia overexpressing Nurrl gene and NSCs co-culture were better than the simple microglia and NSCs co-cultured group.ConclusionNurrl gene not only inhibits activation and inflammatory cytokine production associated microglia, but also promote the secretion of GDNF, BDNF, PDNF for DA neuronal survival and maturation related neurotrophic factors. The microglial of Nurrl gene overexpression and neural stem cells co-cultured can facilitate neural stem cells differentiate into dopamine neurons. Nurrl gene is expected to become one of important and potential targets of regulation for cell transplantation in the treatment of PD.