Study Of The Expression Of S-100β And The Vulnerability During The Oligodenrocyte Lineage Cells Development

The O-2A progenitor cells also maintains the potential to differentiate into either an oligodendrocyte or type 2 astrocyte.From O-2A progenitor cells to mature oligodendrocyte (OL)accompanied with some antigens disappeared or appeared,we investigate the expression of S-100βin the oligodendrocyte lineage cells.S-100βis a Ca²⁺binding protein and widely distributed and conserved in the nervous system of vertebrates.Its accumulation in the brain of mammals may participate in processes underlying the differentiation of individual cell types in the CNS.On the base separation and purification of O-2A progenitor cells,we investigate the expression of S-100βin the oligodendrocyte lineage cells.Periventricular leukomalacia(PVL)is a white matter lesion occurring primarily in preterm infants.Damage to developing oligodendrocyte lineage cell is the hallmark of PVL.The proinflammatory cytokine TNF-αhas been reported to play a role in PVL in both clinical and animal studies,so it is important to study its effects on developing oligodendrocyte lineage cells.Part One The expression of S-100βduring the oligodendrocyte lineage cells developmentObjective To investigate the expression of S-100βin the oligodendrocyte lineage cells,which stage did the S-100βbegain to express.Methods Based on the differential properties of developmental time-course and cellular adhesions,O-2A progenitor cells were achieved by mechanical shaking and differential adhesion method.Oligodendrocyte lineage cells were obtained by using the growth factors and characterized them with cell-specific antigens.With the double immunofluorescence staining,the expression of S-100βin the oligodendrocyte lineage cells were detected. Results O-2A progenitor cells were cultured in the presence of platelet-derived growth factor(PDGF,10 ng/ml)and basic fibroblast growth factor(bFGF,10 ng/ml)for 48 hours,they did not express S-100β.When O-2A progenitor cells were shifted to culture medium with triiodothyronine(T3,30 ng/ml)for 24 hours,they were S-100βpositive.Prooligodendrocyte and oligodendrocytes were also characterized by S-100βimmunoreactivity.Conclusion The beginning expression of S-100βwas between O-2A progenitor and pro-oligodendrocyte and may be correlate with the transition from the dividing bipotential stage to the morphological differentiated.Part Two Study of the vulnerability during the oligodendrocyte lineage cells developmentObjective Used highly purified O-2A progenitor,pro-oligodendrocyte,immature oligodendrocyte to study systematically the vulnerability of tumour necrosis factor-a (TNF-a)on developing oligodendrocyte lineage cells in culture.Methods(1)oligodendrocyte lineage cells at three developmental stages were exposed to increasing concentrations(ranging from 5 to 500 ng/ml)of TNF-a for 48 h,and then viability of cells was determined with MTT assay.cell survival was calculated as the percentage of treatment(OD)/control(OD).(2)oligodendrocyte lineage cells at three developmental stages were exposed to 50 ng/ml TNF-a for 12,24,and 48 h,and then viability of cells was determined with MTT assay.cell survival was calculated as the percentage of treatment(OD)/control(OD).(3)50 ng/ml TNF-a were added to oligodendrocyte lineage cells at three developmental stages for 48 h,In detection of apoptosis was performed using immuocytochemistry of caspase-3.Results(1)TNF-a decreased cell survival in both a time- and a dose-dependent manner.With the increase in TNF-a concentration,a significant decrease in cell viability was observed at all three developmental stages.The survival rate for OLs at three developmental stages was reduced with an increase of exposure time.(2)With the increase in TNF-αconcentration,the survival rate of O-2A progenitor cells was decreased compared with immature oligodendrocyte,pro-oligodendrocyte was also sensitive to a higher concentration of TNF-α.O-2A progenitors were the most vulnerable,whereas pro-oligodendrocyte were less sensitive,although significant cell loss was still observed. Immature oligodendrocyte show resistance to TNF-αtreatment.50 ng/ml TNF-αwere added to oligodendrocyte lineage cells at three developmental stages for 48h,there was no difference in the survival rate of O-2A progenitors and pro-oligodendrocyte,but the pro-oligodendrocyte showed vulnerable to TNF-α.(3)oligodendrocyte lineage cells at three developmental stages were exposed to 50 ng/ml TNF-αfor 48 h,Immunocytochemical staining showed that these cells in the nucleus and cytoplasm of a large number of specific brown-yellow coloring.Conclusion Oligodendrocyte lineage cells were maturation-dependent vulnerability to TNF-α;activated caspase-3 participated in O-2A progenitors,pro-oligodendrocyte and immature oligodendrocyte apoptosis following TNF-αexposure.