A Study Of Nurr1 And Brn4 Gene Transfection On The Controlled Differentiation Of Neural Stem Cells Into Dopaminergic Neurons

PartⅠThe effects on the differentiation of neural stem cells into dopaminergic neurons by Nurr1 and Brn4 gene transfectionObjective:To observe the effects of Nurr1 and Brn4 gene transfection in vitro on the dopaminergic neuronal differentiation and maturation of neural stem cells derived from rat midbrain.Methods:1. Nurr1 and Brn4 full length cDNA were cloned from rat brain tissue by RT-PCR. After digestion, the cDNA were connected into pEGFP-N1 and pDsRed-N1 plasmid vectors respectively, and the recombinant pEGFP-N1-Nurr1 and pDsRed-N1-Brn4 were constructed on the processing of conversion, screening and verification.2. Neural stem cells were isolated and cultured from E14 rat midbrain using serum-free culturing technology. Immunofluorescence detection of Nestin and BrdU were performed to verify the embryonic characteristics and self-proliferating ability of these cells. And the expressions of MAP-2, GFAP, CNP (the distinctive marker for neuron, astrocyte and oligodendrocyte respecitvely) were detected by immunofluorescence labeling to verify their differential multipotent.3. Neural stem cells were divided into 4 groups for transfection, control group was transfected with pEGFP-N1; Brn4 group was transfected with recombinant pDsRed-N1-Brn4; Nurr1 group was transfected with recombinant pEGFP-N1-Nurr1; Nurr1+Brn4 group was co-transfected with recombinant pEGFP-N1-Nurr1 and pDsRed-N1-Brn-4. And all the transfected cells were induced to differentiate in vitro. The transfection efficiency was evaluated by the expression of fluorescin reportergenes, and the effectiveness of transgene expression was determined by Western blot and Real-time PCR. After 3 weeks’culturing, TH/MAP-2 and TH/DAT double-immunostaining were performed to detect the dopaminergic neuronal differentiation of neural stem cells. The areas and perimeters of TH positive cells were measured, and the DA release in the differentiational medium was analyzed using HPLC.Results:1. The full lenth of Nurr1 and Brn4 genes were cloned from rat brain, and the recombinants pEGFP-N1-Nurr1 and pDsRed-N1-Brn4 were obtained through genic recombination.2. Single cell suspensions formed floating neurospheres containing hundreds of cells after one week’s culturing. The neurosphers exhibited both Nestin and BrdU positive, and the differentiated cells expressed MAP-2, GFAP and CNP respectively, the number of GFAP positive astrocytes were the most, and the MAP-2 positive neurons second, but the CNP positive oligodendrocytes were scarce.3. Six to eight hours after transfection, the expression of fluorescin could be observed under a fluorescence microscope, the fluorescin expression increased over time and reached its peak at 48 hours, subsequently, it reduced gradually and disappeared on the whole at about 3 weeks. Image processing showed the transfection efficiency was about 35%, and co-transfection of two different plasmids did not cause competitive inhibition.4. Western blot and Real-time PCR showed that the expression of target genes increased significantly after transfection, and co-transfection of two plasmids did not cause superimposed expressions of Nurr1 and Brn4.5. Immunofluorescent results showed that there were a few TH positive neurons in control and Brn4 groups, the differential rate was about 3%, and the morphology of these cells was immature with small somas and short processes, TH positive cells seldom co-expressed DAT; The number of TH positive neurons increased obviously in Nurr1 group, and the differential rate reached 16.5%. But these cells were also morphologically immature with short neurite extensions, and there were a few TH positive cells that co-expressed DAT; The number of TH positive neurons were the most in Nurr1+Brn4 group, the differential rate was 17%. Morever, these cells exhibited maturer morphology with larger cell bodies and extensive neurite fibers, and the majority of TH positive cells co-expressed DAT.6.HPLC analysis revealed that the differentiational media contained low level of DA in control and Brn4 groups. While DA level in the culture medium increased in Nurr1 group, and was highest in Nurr1+Brn4 group with a concentration of 3.40μg/ml.Conclusions:1. The neural stem cells isolated from rat fetal mescencephalon possess the ability in proliferating and self-renewing, and have multipotential ability to differentiate into neurons, astrocytes and oligodendrocytes.2. Overexpression of Nurr1 could promote the differentiation of neural stem cells into TH immunopositive dopaminergic neurons, but these neurons were immature; Brn4 could not induce the dopaminergic neuronal differentiation of neural stem cells directly, but co-transduction of Brn4 with Nurr1 would improve the neural stem cells to differentiate into morphologically, phenotypically, and functionally mature dopaminergic neurons.PartⅡThe experimental study on the transplantation of transgenic neural stem cells for treatment of Parkinson’s diseaseObjective:To investigate the survival, differentiation and functional exertion of transgenic neural stem cells transplanted into the denervated striatum of PD rats.Methods:1. The unilateral PD rats were prepared by the rat brain stereotaxic technology using single-point injection of 6-OHDA into the right medial forebrain bundle. And APO-induced rotation test was performed to verify the success of PD rats, Nissl staning and TH immunohistochemistry of brain histological section were carried out to verify its reliability.2. The successful PD rats were divided into 4 groups for transplantation, sham group did not transplant any neural stem cells; NSCs group was transplanted with untransfected neural stem cells; Nurr1 group was transplanted with neural stem cells transfected with pEGFP-N1-Nurr1; Nurr1+Brn4 group was transplanted with neural stem cells co-transfected with pEGFP-N1-Nurr1 and pDsRed-N1-Brn4. All the NSCs were labled with DIL and transplanted in the stereotaxic instrument. APO-induced rotation test was performed to observe the behaviour amelioration after transplantation. Twelve weeks later, TH immunofluorescence was carried out to detect the dopaminergic neurons differentiated from transplanted cells, and the DA content in the transplanted striatum was analyzed by HPLC.Results:1. PD rats appear typical rotations, and there were 61% of the rats with the ipsillateral rotations exceeding 210 rotations in 30 minutes. The speed of rotations didn’t decrease over time after constantly observation in 20 weeks; Nissl staining showed that the number of neurons in the substantia nigra pars compacta decreased sharply in the lesioned side. TH immunohistochemistry showed that most TH positive cells in the substantia nigra pars compacta of the lesioned side were lost, the TH reactivity in the striatum of lesioned side was reduced and the TH positive fibers were decreased significantly.2. The behaviour of PD rats did not ameliorate in sham and NSCs groups, rotations increased gradually over time and reached a plateau 10 weeks after transplantation; In Nurr1 group, the rotations of PD rats did not increase any more, and began to decrease at the sixth week after transplantation; The behaviour of PD rats ameliorated obviously in Nurr1+Brn4 group, rotations reduced gradually over time, but it did not reach the normal level yet at the end of 12th week.3. Immunofluorescent results showed that DIL positive cells could be observed in NSCs group, but few of them exhibited TH positive; There were many DIL/TH double-labeled neurons with short neurite extensions in Nurr1 group; The number of DIL/TH positive neurons were the most in Nurr1+Brn4 group, and these cells were morphologically maturer with extensive neurite processes.4. The DA level in the striatum was low in sham and NSCs groups, but it increased significantly in other two groups and reached 7.33μg/ml in Nurr1+Brn4 group, though it was still lower than normal level.Conclusions:Overexpression of Nurr1 could induce the differentiation of neural stem cells into TH positive dopaminergic neurons in vivo, raise DA level in the striatum to a certain extent, and ameliorate the behavioral function of PD rats; Brn4 could improve the survival of dopaminergic neurons in transplantation area. Transplantion of neural stem cells co-transduced with Nurr1 and Brn4 could produce more TH positive dopaminergic neurons, raise DA level in the striatum obviously and ameliorate the behavioral function of PD rats effectively.