Establishment Of PVL Cell Model And Influence On The Proliferation And Differentiation In CG4 After Oxygen-glucose Deprivation

The incidence of brain injury and the neurodevelopmental problems of varying degrees in premature infants are increasing with the growing of premature birth every year. Periventricular leukomalacia (PVL) has become the most important type of brain damage in preterm infants. PVL refers to hypoxia and ischemic injury in periventricular white matter and that is one of causes in children cerebral palsy. The mainly of clinical presentation includes cerebral palsy, epilepsy, mental retardation, language barriers and visually impaired and that influence the quality of life in child. The clinical treatment of PVL is deficient in the effective therapeutic methods so far, mainly due to the pathogenesis of PVL is not very clear. Therefore, in-depth study of the pathogenesis of PVL to guide clinical treatment has become one of the important problems.Oligodendrocyte is type of myelin-forming cell in central nervous system and is highly sensitive to hypoxic-ischemic injury and in brain white matter. Moreover, oligodendrocyte has maturation-dependent vulnerability obviously. It was identified that oligodendrocyte is the key target cell and play a key role during the course of PVL. Based on the above, the establishment of PVL cell model is necessary for explore pathogenesis of PVL.Olig transcription factor belongs to the bHLH transcription factor family, including olig1 and olig2, that are two novel bHLH proteins which appear to be specifically expressed in oligodendrocyte progenitor throughout the CNS. Olig1 has function on spinal cord patterning and on maturation of oligodendrocytes. The function of Olig2 is necessary for oligodendrocyte progenitor cells development. Olig gene decides the proliferation, specification, and differentiation of cortical progenitor cells. However, little is known about the involvement of bHLH Olig in oligodendrocyte progenitor cells (OPCs) development and injury. Hypoxia-inducible factor 1 (HIF-1) is also a basic-helix-loop-helix transcription factor that plays essential roles in animal development and physiology. HIF-1αhas been reported to suppress apoptosis in hypoxia condition and mediate adaptive responses to reduced oxygen availability. Moreover, another type of transcriptional factor Nkx2.2 regulates the differentiation and maturation of oligodendrocyte. Nkx2.2 and other relevant transcriptional factors co-operated to participate in differentiation of oligodendrocyte.CG4 cells were selected in order to establish PVL cell model instead of primary oligodendrocyte precursor cells which is closely related with the occurrence of PVL. Cell death rate, proliferation and apoptotic proteins ratio were selected to evaluate PVL cell model of hypoxia, oxygen-glucose deprivation (OGD), OGD and reoxygenation treatment and cell morphology, specific protein expression, proliferation level and relative Olig transcription factors expression which are correlated with differentiation and development were examined to observe the influence of oxygen-glucose deprivation. In addition to, Olig transcription factors and relative transcription factor were evaluated to observe the influence of these transcription factors on CG4 cell differentiation and development by establishing OGD and differentiation model and the relationship of Olig transcription factor and ERK pathway were further studied.This study was completed in Neuropsychiatric Research Institute, University of Manitoba, Canada. The following are the details of methodology and major results obtained in this study.1. The selection and evaluation of CG4 cellsCG4 cell line, the genetically modified product, derived from the primary culture of neonatal rat cortex and developed from the bipotential oligodendrocyte-type 2-astrocyte progenitor. CG4 cell is a permanent cell line which can be passage indefinitely. CG4 cells were obtained from Neuropsychiatric Research Institute, University of Sascatchewan, Canada. (1) The morphology of CG4 cells were observed by microscope: the morphology of CG4 cells and oligodendrocyte precursor cells are similar; their morphology are similar to immature oligodendrocytes and mature oligodendrocytes after induced differentiation(2) Specific proteins expression of CG4 cells were observed by fluorescence microscope: the PDGFRαprotein which is the marker of oligodendrocyte progenitor cell was expressed in CG4 cells and mainly located in the nuclear surface of cells. The CNPase protein which is the marker of immature oligodendrocytes and mature oligodendrocyte was expressed in CG4 cells and located at the cytoplasm of CG4 cells after differentiation treatment.2. Establishment of cell model(1) Establishment and evaluation of hypoxia alone cell model: CG4 cells were incubated with 6, 12, 24, 36, 48, 60, 72 hr in the CO2 hypoxic incubator and cell death rate, proliferation level and apoptotic proteins were examined. The results showed that the cell death rate was decreased significantly with the extension of hypoxic time. The cell death rate in each hypoxic group was lower than 10%; the cell proliferation level was increased significantly with extension of hypoxic time. Whereas, the cell proliferation level was decreased significantly in hypoxic groups comparing with the normal groups; the apoptotic proteins Bcl-2/Bax ratio was increased firstly and then reduced with the extension of hypoxic time.(2) Establishment and evaluation of OGD cell model: the medium of CG4 cells was changed into the glucose-free medium and following incubated with 6, 12, 24, 36, 48, 60, 72 hr in the CO2 hypoxic incubator and cell death rate, cell proliferation level, apoptotic proteins expression were examined. The results showed that the cell death rate was decreased significantly with the extension of OGD time. The cell death rate in OGD 6, 12, 24, 36hr is higher than 10%; the cell proliferation level was increased significantly with extension of OGD time; the apoptotic proteins Bcl-2/Bax ratio was increased firstly and then reduced with the extension of OGD time. (3) Establishment and evaluation of OGD and reoxygenation cell model: the medium of CG4 cells was changed into the glucose-free medium and following incubated with 24 hr in the CO2 hypoxic incubator following incubated with 24, 48, 72, 96, 120 hr in the normal CO2 incubator and cell death rate, cell proliferation level, apoptotic proteins expression were examined. The results showed that cell death rate in OGD and reoxygenation is higher markedly than 10%; the cell proliferation level was inhibited significantly by OGD and reoxygenation comparing with the normal group; the apoptotic proteins Bcl-2/Bax ratio was decreased significantly comparing with the normal group.4. The influence of OGD on CG4 cells(1) OGD affects the CG4 cell death and proliferation: the CG4 cell death rate and proliferation level were examined after treatment with OGD 6, 12, 24 hr. The results showed that the cell death rate in OGD 6, 12, 24 hr is higher than 10%; the cell proliferation level was increased significantly with extension of OGD time; the proliferation level was decreased significantly in OGD groups comparing with the normal groups especially in OGD 24hr.(2) OGD regulates related proteins expression in CG4 cells: the influence of OGD on related proteins expression was evaluated by Western blotting and immunofluorescence staining techniques. The results of Western blot: After OGD 6, 12, 24 hr treatment, Olig1 and Olig2 expression were decreased significantly comparing with the normal group. HIF-1αand Nkx2.2 expression were increased with OGD 6 hr and then decreased with OGD 12, 24 hr. PDGFRαexpression was decreased by OGD; The results of immunofluorescence staining: After treatment with OGD 6, 12, 24 hr, Olig1 expression is markedly located in cytoplasm and the Olig1+ CG4 cell number was decreased comparing with the normal group; Olig2 is located in the cell nuclear and the Olig2+ CG4 cell number was decreased with OGD 12, 24 hr except 6 hr. Nkx2.2 is located in cytoplasm and Nkx2.2+ CG4 number was decreased with OGD 12, 24 hr except 6 hr.5. The influence of OGD and differentiation on CG4 cells (1) Cell proliferation level: CG4 cell was treated with OGD 6, 12, 24 hr and following differentiation 3 days, the results showed that cell proliferation was not inhibited by OGD 6 hr and differentiation but was increased with OGD 12, 24 hr and differentiation.(2) Related proteins expression level: The influence of OGD and differentiation on related proteins expression was evaluated by Western blotting and immunofluorescence staining techniques. The result of Western blotting: Olig1, Olig2, HIF-1αand Nkx2.2 expression were increased significantly after treatment with OGD 6 hr and following differentiation 3 days in CG4 cells and were decreased significantly after OGD 12, 24 hr and following differentiation 3 days. The two specific proteins of oligodendrocyte, PDGFRα/ CNPase expression ratio was decreased first, then increased after OGD and differentiation treatment; the result of immunofluorescence staining: After treatment with OGD 6, 12, 24 hr in CG4 cells, Olig1 and Nkx2.2 expression is located in cytoplasm and Olig2 is located in cell nuclear. Olig1, Olig2和Nkx2.2 was increased on OGD 6 hr and differentiation firstly and then decreased on OGD 12 , 24 hr and differentiation.6. OGD regulates Olig protein expression through ERK pathway possibility(1) The change of p-ERK/ERK protein expression on OGD and OGD following by differentiation treatment in CG4 cells: p-ERK/ERK was decreased significantly on OGD treatment. The results suggested that OGD inhibit ERK expression; p-ERK expression was increased firstly in OGD 6 hr following differentiation and then decreased in OGD 12, 24 hr and differentiation. The results showed OGD and differentiation promotes ERK phosphorylation firstly, and then inhibits ERK phosphorylation.(2) The influence of p-ERK inhibitor on p-ERK/ERK and Olig proteins expression with OGD treatment in CG4 cells: Experiment were divided into normal group, the normal + p-ERK inhibitor groups, OGD and OGD + p-ERK inhibitor groups, the two time points 12, 24 hr were selected. CG4 cells were treated with p-ERK inhibitor for 30min in OGD+inhibitor and normal+inhibitor groups and then OGD and OGD+inhibitor groups were treated with OGD 12, 24 hr, p-ERK/ERK expression were examined using Western blotting. The result showed that inhibitor increases p-ERK activation in normal and OGD groups. The inhibitor can increase Olig1 and Olig2 expression.The conclusions of this study are as follows:1. CG4 cells have the bipolar morphological structure and express specific protein PDGFRαof oligdendrocyte and that suggested CG4 cells possess the character of oligodendrocyte progenitor cells; cell morphology consistent with immature oligodendrocytes and mature oligodendrocytes after treatment with differentiationin CG4 cells and CG4 cells have the stage-specific protein expression, CNPase, indicating that CG4 cells have the capacity for further differentiation and development.2. OGD treatment in CG4 cells can be chose for PVL cell model. Cell death rate, proliferation level and related apoptotic protein Bcl-2/Bax expression were evaluated for selecting the cell model of PVL.3. OGD influences significantly Olig1, Olig2 and Nkx2.2 expression. Olig1, Olig2 and Nkx2.2 expression were up-regulated first, then down-regulated after OGD and differentiation. The results indicated that OGD induces PVL through the influence of OGD on cell maturation and differentiation.4. Inhibition of PDGFRαexpression by OGD treatment suggested the ability of cell proliferation was decreased. PDGFRα/ CNPase expression ratio was decreased first, then increased after OGD and differentiation treatment. The results showed that OGD significantly influenced the differentiation and maturation of CG4 cells.5. The up-regulation first, then down-regulation of HIF-1αexpression in CG4 cells by OGD treatment suggested that OGD induced adaptation reaction of CG4 cells first, and then the adaptation reaction was decresed, Olig gene was down-regulated and cell differentiation retardation with extension of OGD exposure time.6. The change of p-ERK protein expression induced by OGD treatment suggested that the influence of transcriptional factors of Olig1, Olig2, HIF-1αand Nkx2.2 expression by OGD may be through the ERK pathway.This working provided the cell model of PVL in vitro and also provided relevant theoretical and experimental basis for studying the pathogenesis of PVL and clinical treatment.