| Objective:Study of Parkinson’s disease has been the focus of neuroscience research field, neuroscience spend a lot of time to devote themselves to the study of the pathogenesis of Parkinson’s disease, treatment, progress continued to new. In this paper, we study the therapeutic effect of neural stem cell transplantation on Parkinson’s disease rats by in vivo experiments, and explore the microenvironment of transplantation, and provide theoretical basis for clinical application.Methods:1.Neural stem cells isolated, cultured and identified:NSCs isolated from the lateral component of the midbrain of fetal rats at gestational age of 14 or 15 days, by trypsin digestion were made into cell suspension, were cultured in B27, EGF and bFGF, serum free medium. Immunocytochemistry identification of neural stem cells phenotype(Nestin), Changed half medium on time, Cultivated to spare.2. Differentiation of NSCs was detected by immunocytochemistry(TH, beta-3-Tublin, GFAP)3. Rat model of Parkinson’s disease was established and consulted the rat George Paxinos and Charles Watson Atlas (6th Edition)260-280g (279+16.7) male SD rats, 2% pentobarbital sodium (0.6ml/100g) intraperitoneal injection of anesthesia. After successful anesthesia,in the rat brain stereotaxic instrument properly fixed and scalp incision, keep anterior/posterior fontanelle at the same level. According to rat brain stereotaxic atlas,in the right MFB (medial Forebrain bundle):(mm A/P:-3.6; mm L:+2.0; V:-9.0 mm); VTA (ventral tegmental area):(mm A/P:-6.0; mm L:+0.5; V:-8.0 mm); CPu (Caudate Putamen, Striatum):(A/P:+1.2mm; L:+2.5mm; V:-5.0) as stereotactic injection target, injected 4μl 6-OHDA (3μg/μl), to 1μ per min slow injection, after injection stay injector 6 minutes, then slowly withdrawing the needle. After surgery 7 days,began to induce rats to the left side of the rotation, After intraperitoneal injection of APO (0.5mg/kg) 10 minutes, recording of the rotation number, each time 5min.Every 7d, continuous test 6 times, greater than or equal to 7 circle per min as PD rats model.4.mNSCs transplantation:Rat model of Parkinson’s disease were randomized into 2 groups, ①Control group (PBS), ②mNSCs transplantation group. Before transplantation cells collected by high speed centrifugation(1200 rpm/min), with 106/ul cell density injection.Pentobarbital sodium 0.6ml/100g intraperitoneal injection of anesthesia. To be successful anesthesia, Fixed on the stereotaxic apparatus, in the right striatum (A/P:+0.6 mm, mm L:+3.0; V:-5.0 mm),using micro syringe injection of 5μLcell suspension.After cell transplantation, intraperitoneal injection of APO (0.5 mg/kg) induced rat rotation behavior in 14d,28d,42d.5. After mNSCs transplantation in 14d,28d,42d the rats were anesthetized, the brain tissue was taken, and WB, PCR, immunofluorescence staining was used to detect the expression of dopamine neurons related genes and protein.6. Statistical analysis:SPSS 17.0 for statistical processing, data on (x-±S) Statistical test method with the paired t test, single factor variance analysis (one-way AVONA).P>0.05, no statistical difference; P<0.05, had statistical difference; P <0.001, had significant statistical difference.Results:1. In vitro cell culture results showed that neural stem cells were globular suspended growth,part of adherent growth, Nestin identified positive, a large number of cell connections were formed after differentiation, TH, β-3-Tublin, GFAP identified positive.2. PD model induced results:1W vs 2W:P= 0.665> 0.05; 2W vs 3W:P= 0.047< 0.05; 3W vs 4W:P=0.003<0.05;4W vs 1/2W:P<0.001;5W vs 1/2/3/4W:P<0.001;6W vs 1/2/3/4/5W:P<0.001. After neural stem cell transplantation:(1) control group:2W vs 4W:P= 0.003< 0.05;6W vs 2/4W:P<0.001;(2)Neural stem cells transplantation group:2W vs 4W:P=0.011<0.05;2W vs 6W:P<0.001;4W vs 6W:P= 0.177> 0.05.(3) Neural stem cells transplantation group 2W vs control group 2/4/6W:P<0.001; Neural stem cell transplantation for 4W vs control group 2/4/6W:P<0.001;Neural stem cell transplantation 6 W vs control group 2/4/6 W:P< 0.001.3.The number of positive cells in the mNSCs transplantation group was significantly different than the control group, and the number of cells decreased with the time passing. Nestin counting results:2/4/6W vs control group:P<0.001.TH counting results, transplantation group at each time compared with the control group:P<0.001;6W vs 2W:P=0.003<0.05.VMAT2 counting results:2/4/6W vs control group:P<0.001; 6W vs 2W:P= 0.158>0.05. DAT counting results:the control group vs 2W:P=0.015<0.05,other groups compared with each other P<0.001.Pitx3 counting results:the control group vs 2W:P<0.001.Lmx1a counting results:the control group vs 4W:P<0.001;the transplantation group in 4W vs 2/6W:P<0.001. Neural stem cells differentiate into dopaminergic neurons, which accounts for about 7%-24%.4. Western Blot:Compared with the control group, the expression of TH protein in neural stem cell group was increased (P<0.001). In the control group at different time (P<0.001),protein expression was gradually decreased with the time prolongation. neural stem cells transplantation group 4W vs control group at each time point:P< 0.001.neural stem cells at each time point:P< 0.001.5.PCR:mNSCs transplantation 4W vs other time point P>0.001; Control group every time point Statistical analysis P>0.05.Conclusion:Neural stem cell transplantation can improve Parkinson’s disease rat behavior. Transplanted neural stem cells are capable of differentiation, colonization, and migration, but the number of dopaminergic neurons is less, and it is not stable and effective long-term survival, and it faces many challenges. |
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