Dynamic Changes Of LINGO-1 In Rat WMI Model And Pilot Study On WMI Treatment With OPCs With Low LINGO-1 Expression

BackgroudMatter injury white (WMI) is a common type of brain injury in preterm infants, and it is also one of the main causes of cerebral palsy and mental retardation in children. The survival rate of premature infants has greatly improved with the development and improvement of neonatal intensive care unit, but there is still a lack of effective treatment for WMI in premature infants at home and overseas, resulted in an increasing number of children with cerebral palsy, cognitive impairment and learning ability in our country every year. The main cause of WMI is the oligodendrocyte progenitor cells (OPCs) injury, which lead to retard or damage of myelin; OPCs are immature oligodendrocytes which are able to proliferate, migrate, and finally differentiate into mature OL and form myelin structure. WMI patients lost a large number of OPCs and lead to the emergence of myelin disorder, OPCs transplantation is the most effective and direct way to promote the myelin sheath regeneration, but mature OL is not suitable to be used as seed cells for transplantation because of the loss of migration ability. OPCs are regarded as the best seed cells resource for WMI treatment. Although OPCs has brought hope for the treatment of patients with WMI, the transplant treatment still faces a lot of problems. As a foreign matte, the micro environment is very important for transplanted OPCs. Recent studies have found that a variety of negative regulators have an important effect on their differentiation and survival. As a typical representative, LINGO-1 is a transmembrane protein composed of 614 amino acids, which is specifically expressed in the central nervous system neurons and glial cells. Animal experiments have found that LINGO-1 plays an important role in the regulation of OPCs differentiation and myelin formation and which is a potent negative regulator. Downregulation of LINGO-1 expression is essential for myelin formation in vivo.In summary, the key to the treatment of premature infants with WMI is to promote the regeneration of the myelin sheath. At present, the main problem that affects the regeneration of the myelin sheath is the survival and differentiation efficiency of exogenous implanted OPCs. LINGO-1 is one of the key factors that may break through this bottleneck problem. The purpose of this paper is to establish a model of 3-day-old neonatal SD white matter injury by hypoxia ischemia, and to explore the dynamic change of LINGO-1. Construct the effective LINGO-1 specific siRNA lentivirus vector. The human OPCs with low expression of LINGO-1 transplanted into the WMI model, the survival, migration and differentiation of transplanted cells to be explored after implantation.Chapter 1 the dynamic changes of LINGO-1 in a 3-day-old neonatal SD rat WMI model modelObjective To explore the dynamic changes of LINGO-1 by establishing a model of ischemic white matter injury in 3-day-old neonatal SD ratMethods Neonatal rats of 3d were divided into two groups randomly, model group (n=42), sham operation group (n=42), a model of cerebral white matter injury was established by ligation of the right common carotid artery combined with hypoxia, and the rat of sham operation group was only separated from the right common carotid artery without ligation. Two groups were furtherly divided into six groups according to the different time pionts (1d,3d,7d,14d,21d and 28d). Six rats in WMI group and sham group were randomly selected for modeling and identification. The pathological changes,immunofluorescence staining of MBP and early motor dysfunction assessment were used to evaluate the severity of the model. Dynamic expression of LINGO-1 mRNA and protein in different time pionts were analyzed.Results1. Hematoxylin eosin (HE) stainingIn group WMI, the total cortical white matter cells were swollen, sparse, the gap widened, the arrangement disorder, and the small infarction foci were found, which showed a selective damage. Staining results were similar to pathological features of preterm infants with WMI.2. MBP expression in immunohistochemical stainingIn group WMI, there was a significant loss of myelin sheath in the white matter area of the surgical side, and there was no obvious loss of myelin sheath in the white matter of the Sham group.3. Dynamic expression of Lingo-1mRNA in different time pointsThe results of qPCR show that one day after the model was made, LINGO-1 mRNA expression is significantly increased (3.79±0.30), seven days later the expression reaches its peak (6.64±0.25). Since then show a downward trend, the expression of WMI group was almost close to sham group at the time of 28d, p>0.05. Western blot results also get the same conclusion.4. Early motor dysfunction assessmentThe result show that mild motor dysfunction occurred in the injured side of WMI group rats, the difference of score between WMI group and sham group had statistical significance, p<0.05.ConclusionsThe model was successfully established, and the pathological and behavioral changes were similar to the clinical features of white matter damage in premature infants.The study found that the LINGO-1 protein was regularly and pathologically increase.Seven days late the expression reaches its peak.Chapter 2 Construction and selection of effective of shRNA lentiviral vector targeting human LINGO-1Objective The purpose of this study was to construct a short hairpin RNA (shRNA) interference lentiviral vector targeting the human LINGO-1 gene and to evaluate its inhibitory effect, screening for the best interference efficiency of the lentiviral vector.Methods The 3 pairs of LINGO-1 shRNA sequence was designed and synthesized according to the sequence reported in GenBank, corresponding shRNA1, shRNA2, shRNA3 group and a negative control sequence shRNA4 group (no targeting sequence of random scatter). The recombinant plasmid vector was sequenced to verify whether the recombinant vector sequence was identical with that of the synthetic interference vector; the correct plasmid vector was packaged into the lentiviral vector in 293T cells and the virus titer of each group was determined. After infecting the human glioblastoma cells with the packaged lentiviruses, we analyzed the inhibitory effect by immunofluorescence, qPCR and Western blot respectively.Results1. Recombinant vector sequencing, packaging and titer determinationGene sequencing confirmed that four LINGO-1 recombinant vectors were successfully constructed, the lentiviruses were packaged in the transfected 293T cells, with the final viral content of shRNAl=4E+8TU/ML, shRNA2=2E+8TU/ ML, shRNA3=1E+8TU/ML and shRNA4=3E+8TU/ML.2. Immunofluorescence staining resultsU251 cells were infected by virus, the positive cells of GFP was more than 96%.The expression of RNA3 group cells showed a weakening phenomenon than other groups, which showed that its interference efficiency is effective.3. qPCR resultsQuantitative PCR showed that the relative expression of LINGO-1 gene mRNA in U251 cells infected by the lentiviral vector was that shRNAl group=0.88±0.03, shRNA2 group=0.31±0.02, shRNA3 group=0.18±0.03, shRNA4 group=1.00±0.00; The relative expression of LINGO-1 gene mRNA in shRNA3 group was significantly lower than that in shRNA4 group, the difference was statistically significant, it means that the mRNA group shRNA3 decreased by 82% compared to the shRNA4 group.4. Western blot resultsAnalysis of the gray-grade scanning show that the expression of LINGO-1 protein in each group was significantly lower, which compared with the control group, and the expression of shRNA3 group (shRNA3=0.28±0.03) protein was the least, p<0.01.ConclusionsLentiviral shRNA interference vectors targeting human LINGO-1 were constructed successfully, which could suppress the expression of the human LINGO-1 gene. Our recombined lentivirus vector will provide a basis for furture study of the role of LINGO-1 gene in axon regeneration the LINGO-1 gene.Chapter 3 Study on white matter damage model after the low expression LINGO-1 transplanted into the brainObjective To explore the survival, migration and differentiation of low expression LINGO-1 human OPCs transplanted into WMI model.Methods Human OPCs was induced by neural stem cells, and OPCs specific markers were identified by immunofluorescence staining. High purity OPCs were transfected by ShRNA3 sequence lentiviral vector (MOI=10), calculate the number of GFP positive cells of OPCs.The LINGO-1 expression was detected by qPCR, and the interference efficiency was verified. After transfection, OPCs was transplanted into WMI model 7 days later. After 1,2 and 4 weeks of transplantation, the frozen sections were made to observe the survival, migration and differentiation of transplanted cells. After 4 weeks of transplantation, according to the survival of cells, the MBP staining was performed to evaluate the improvement of the white matter.Results1. Human OPCs was induced by neural stem cellsHuman OPCs was induced by neural stem cells, and OPCs specific markers were identified by immunofluorescence staining, the result show that the expression of PDGFR,04 and Sox10 were high expressed in cells.Only a very small number of cells expressed astrocytes marker GFAP and neuronal marker Tuj-1.2. Transfection and interference efficiency of OPCsHigh purity OPCs were transfected by shRNA3 sequence lentiviral vector, the number of GFP positive cells was more than 98%. PCR results showed that the RNA3 sequence of the lentiviruses can effectively reduce the OPCs LINGO-1 expression, and the interference efficiency is 84%.3. State of OPCs after transplantationAfter transplantation 1 week, the cells of OPCs transplant group have survived in the host brain tissue, cells mainly focus on the injection hole position; the same to control group of OPCs transplantation. Two groups of transplanted cells did not have obvious migration. After transplantation 2 weeks, cells are still around the injection needle, but the number of cells decreased without moving. The control group of OPCs transplantation was significantly reduced. After transplantation 4 weeks, GFP labeled OPCs cells were not found.ConclusionsLentiviral shRNA interference vectors targeting human LINGO-1 were constructed successfully.which could suppress the expression of the human LINGO-1 gene of OPCs. After transplantation, the low expression LINGO-1 of OPCs can survive in a short period of time, but there is no significant migration and differentiation in the long-term.