A Study Of The Effects Of NOV Gene On The Development And Differentiation Of Oligodendrocyte Precursor Cells And Transplantation On Hypoxia Ischemia In Rats

Oligodendrocyte progenitor cells (OPCs) have the capability of self-renewal and unsymmetrical differentiation in vitro, which are identified as multi-potent progenitor cells or intrinsic CNS adult stem cells. OPCs express several markers, such as NG2, the platelet-derived growth factor-a receptor (PDGFaR) and 04. Thus far, studies have provided abundant evidence that OPCs are highly reactive cells that respond to several types of nerve injuries, such as viral infection, immune reaction, nerve degenerative disease, CO-induced demyelination, dysbolism and Hypoxic-ischemic encephalopathy. OPCs belonged to oligodendrocyte lineage. But if the only function of OPCs was differentiated into oligodendrocyte? OPCs was upregulated as a result of physical, viral, excitotoxic and inflammatory insults to the CNS. Following demyelination OPCs number increaseed in the immediate vicinity of the lesion and rapid remyelination ensues, which was different from astrocyte and microglia. The number and immune reaction of OPCs increased after nerve degenerative disease. The direct evidence that OPCs can repair the damage and promote myelogeny was poor. The study of cytobiology and electrophysiological characteristics of OPCs were deficiency too. But the effects of OPCs on the development, differentiation and regenerationon in central nervous system was not clear.Perinatal hypoxia-ischemia (HI) is a significant primary cause of brain damage of neonates. Periventricular white matter injury (PWMI) is the predominant form of brain injury underlying this neurological morbidity. The spectrum of chronic PWMI includes focal cystic necrotic lesions (periventricular leukomalacia; PVL) and focal, multifocal, or diffuses myelination disturbances. Despite major advances in obstetric and perinatal medicine, there has been little chang in the incidence of hypoxic-ischemic encephalopathy, which has been estimated to occur in approximately 3-4 neonates per 1000 live-born at term, the incidence is higher in small preterm neonates. Depending on the gestational age and the severity of the insult, between 10% and 60% of asphyxiated neonates who have hypoxic-ischemic encephalopathy expire during the neonatal period and 25% or more of the survivors develop permanent neuropsychological deficits such as cerebral palsy, mental retardation and epilepsy. OPCs have been demonstrated to be intrinsically more vulnerable to injury than mature oligodendrocytes under conditions of oxidative stress, oxygen glucose deprivation (OGD) in vitro, hypoxia-ischemia in vivo (Back et al.,2002), and glutamate receptor-mediated excitotoxicity. Perinatal hypoxia-ischemia (HI) is a significant primary cause of PWMI. The fact that OPCs appear coinciding with the high-risk period for PWMI and is selectively targeted by HI suggests that OPCs are the major target cell of injury in PWMI. However, Our understanding of the fundamental mechanisms involved in selective injury of OPCs to HI in the developing brain is still limited.Nephroblastoma Overexpression gene, abbreviated as NOV, is a proto-oncogene, which was discovered in 1991. NOV protein, which is encoded by NOV gene, is a kind of insulin-like growth factor (IGF) binding protein (IGFBP), and one of the families of IGF. NOV gene is a multipurpose factor that regulates cell proliferation. IGF-I was necessary for the proliferation of the nerve stem cells, which could affect the nerve stem cells through tyrosine protein kinase pathway. bFGF and EGF couldn't stimulate the proliferation of the nerve stem cells. The effect of NOV on the development and differentiation of OPCs is unknow. Marian Zaka and Shuying Lin found that IGF-1 could promote the proliferation of OPCs and decrease the apoptosis of OPCs induced by Toxic substance. Since NOV was the important regulating factor of nerve system development and regeneration, and IGF was the encode binding protein, we want to see if the NOV have the effects on promoting the regeneration and function recovery of OPCs after OGD. Therefore, this investigate is divided into three part:Part 1:We use PDGFaR antibody to analyze the developmental regulation of PDGFaR+ cells in the normal postnatal rats CNS from birth to old age, including the morphology, distribution, growth characteristics and the quantity of PDGFaR+ cells population. Meantime, double labelling with antibodies against PDGFaR and NG2 is applied and the results compare to the different developmental stage. It is expected these data will help us to find out the developmental regulation of the PDGFaR+ OPCs and compare to the NG2+ OPCs in the CNS as well as the primary significance of co-localization to the OPCs developmental lineage.The main results are as following:1. The morphology, distribution, electrophysiological characteristics and developmental regulation of PDGFaR+ cells in rat CNS are similar to that of NG2+ cells.2. The number of PDGFαR+ cells at early developmental stages is much more than that at later stages. Specifically at the later postnatal stages,25% of the NG2+ cells do not express PDGFαR and the morphology of PDGFαR-/NG2+ cells is similar to that of NG2+ cells.3. Aging has an effect on the morphological feature, number and developmental regulation of OPCs in rat CNS.In conclusion, both PDGFαR and NG2 are markers of developing OPCs. PDGFαR expression is seen on the most developing and immature precursors, then the cells acquiring NG2 expression as they mature. At the preoligodendrocyte stage, PDGFαR expression has ceased, whereas NG2 expression persists, at least at the beginning of this stage. NG2+ cells may be a mature glial cell type distinct from conventional OPCs.Part 2:To establish an oxygen/glucose derived(OGD) model of SD rat's oligodendrocyte precursor cells in a nomaxic environment in vitro with dithionite sodium and DMEM without glucose, and evaluate the model by investigating to the changes of cellular shapes and survival caused by OGD. And 4-day-old SD rats were anesthetized and subjected to a hypoxic/ischemic injury obtained by combination of monolateral carotid ligation and exposure to 8% oxygen for 2h. Histopathologic changes and the express of MBP and PDGFαR were observed to evaluate the model. The main results are as following:1. When the PDGFaR positive OPCs were cultured with sodium dithonite and DMEM without glucose, the morphologic changes were obvious:cell body swelling and the processes swelling, deformed and collapsed gradually. The survival rate decreases gradually with the time and the rate was about 50% at 60 min while it decreased to a level lower than 50% at 90min.2. After a hypoxic/ischemic injury obtained by combination of monolateral carotid ligation and exposure to 8% oxygen for 2h, the histological damage was observed and the numbers of the apoptotic nerve cells increased. The express of MBP,NF200 and PDGFαR decreased.The second part revealed that DMEM without glucose and sodium dithionite can be applied to the construction of GOD model in PDGFaR positive oligodendrocytes in a normoxic incubator in vitro. The damages of oligodendrocyte precursor cells caused by hypoxia-ischemia in vitro can be analogized with this model, and similar damages occur in the oligodendrocyte precursor cell in vitro.Part 3:We built pEGFP-N 1 vector containing NOV gene complete sequence to transfer, and examined its expression by immunohistochemisty and conditioned medium (CM) from cultured NOV/COS-7 cells (NOV-CM) was collected. The effects of NOV on the proliferation and differentiation of OPCs were studied and see if the COS7s engineered by NOV gene have the effects of promoting the regeneration and function recovery of nerve injury.The main results are as following:1. The NOV gene complete sequence were synthesized by RT-PCR, and ligated into EcoR I and Hind III sites of pEGFP-Nl vector. The nucleotide sequences of the cDNA were determined. The vector was led into cultured COS-7 cells using DOTAP liposomal transfection reagent. The expression of NOV protein was detected by Western blot and immunocytochemistry.2. We built pEGFP-N1 vector containing NOV gene complete sequence and conditioned medium (CM) from cultured NOV/COS-7 cells (NOV-CM) was collected. NOV-CM was treated to the OPCs of OGD. Results showed NOV gene promoted the survival of OPCs and the recovery of structure and function of neuron and myelin after injury. Our results indicated that NOV gene probably involved in the processs of repair of nerve injury and rebuilding of function. The results of this study provide some fundmental information for the clinical application of OGD.