The Effect Of Erythropoietin And Methylprednisolone In Combination On Spinal Cord Edema

The first part Culture and identification of spinal astrocytes from ratsObjectives To culture and identify primary astrocytes in spinal cord of neonatal rat.Methods The spinal cord were isolated from neonatal rats born within 3 days,cells were inoculated in 75cm2 cell culture flask by 107?109/L.The flasks were placed in the incubator(5%CO2,37?).Cells were cultivated by DMEM/F12 medium was added to 10%fetal bovine serum for 2 weeks.The astrocytes were purified by differential velocity adherent technique,swinging and passaging.We will observe the morphological change of the cells,identify the astrocytes by immunofluorescence staining.Results Astrocytes of high pirity can be achieved by shaking andpassaging.The cells are positive to GFAP staining.GFAP-positive cells accounted for more than 98%of the total cell count.Conclusion Primary culture astrocytes canbe get by newborn rat spinal cord.High-purity astrocytes can be obtained by passaging and differential velocity adherent technique.The second part The establishment of rat astrocytes injury modelObjectives To establish the spinal cord ischemia model of rat astrocytes and observe the biological change.To investigate the effect of EPO combined with MPSS on astrocytes cultured in vitro,and to explore the mechanism of its effect on spinal cord ischemia and edema.Methods The astrocyes were isolated from spinal cord of neonatal rats born within 3 days,inoculated in the 0.01%poly-L-lysine-coated bottle by 107?109/L,incubator(5%C02,37?),cultivated by DMEM/F12 media added with 10%fetal bovine serum Then placed in the incubator(99%Ar,0.01%O2,37?),cultivated by DMEM media without fetal bovine serum for 12h.Make injury model by OGD4h/R12h to observe cell morphological changes before and after injury.Cell proliferation activity of astrocyte was analysed by MTT assay.The damage degree of the leakage rate of eell melbrane Was analysed by LDH assay.Flow cytometer analysis the expression of apoptosis and related protein expression of apoptosis injury rate.AQP4 mRNA levels of astrocytes analysed by PCR assay.Expression levels of AQP4 protein of astrocytes analysed by Western blot assay.Establish an ischemic model of astrocyte injury by OGD injury;and then devided to the saline control group,MPSS(10 ug/ml)group,EPO(10 u/ml)group,MPSS(10 ug/ml)and EPO(10 ug/ml)intervention group,cells were collected after intervention respectively in 18 h;observation:1,To observe the effects of different combinations of intervention on the morphology of astrocytes by microscope;2,cell activity was measured by MTT assay,the damage degree of the cell membrane me asured by LDH assay,and detecting the injury rate of apoptosis and analysis the apoptosis related protein(caspase-3)expression;3,astrocytes AQP4 mRNA expression levels was analysed by RT-PCR assay and changes of AQP4 protein analysed by Western blot assay.Results Cells with smaller and shorter cytoplasmic process after OGD/R,the majority of cell with larger and longer cytoplasmic,few of cells death.MTT showed decreased proliferation of astrocytes after injury,EPO alone and saline recovery had no significant effect on cell proliferation,MPSS with or without EPO has significant protection effect on cell proliferation;recovery increased cell proliferative activity and the difference was statistically significant;the damage degree of the leakage rate of cell membrane detectioned by LDH assay and the expression of injury rate of apoptosis and apoptosis related protein,suggesting that damage model was successfully established.PCR result showed:the expression AQP4mRNA was decreased by injury and increased after recovery,there were significant differences,EPO and MPSS combined application of recovery has obvious statistical significance of cell proliferation and apoptosis;single EPO or single MPSS could promote the AQP4 damage of cells increase the expression of mRNA is better than the single drug application;the combination of EPO and MPSS has significant protection effect on the expression of AQP4 mRNA levels;Western blot showed:the expression AQP4 protein levels was decreased after injury,there were signifieant differences.The OGD/R model was successful established.OGD damage can inhibit the proliferation of astrocytes.Drug application promotes the expression of apoptosis related proteins and AQP4 protein,the difference was statistically significant.EPO and MPSS have obviously protection effect to injuried astrocytes.Conclusions The spinal cord ischemia model of rat astrocytes were established.The applying of EPO and MPSS have obviously protection effect for injuried astrocytes.The third part the blocking of protein kinase C signaling pathwayObjective To study the effect of Ro 31-8220 on astrocytes in vitro culture.Method The astrocytes passaging to the 3-4 generation,intervented with PKC pathway inhibitor Ro 31-8220,eells were collected after 0.5h intervention and the expression of AQP4 mRNA levels measured by PCR assay.Western bloting technique was used to detection the expression of AQP4 protein after 12h,the cell activity was measured by MTT colorimetric method,the damage degree of the leakage rate of cell membrane analysis by LDH assay,then detect apoptosis related protein.Results The effects of MPSS on AQP4 could be blocked by the inhibitor Ro 31-8220,and the function of EPO can be enhanced by MPSS.Conclusions The effects of EPO and MPSS were dependent on the protein kinase C(PKC)signaling pathway.