The Experimental Research On Methylprednisolone And Erythropoietin In Combination On Astrocytes In Vitro

The first part Establishment of Primary astrocytes modeling and identification Primary astrocytesObjectives To make primary astrocytes by newborn rat brain , and identificaty astrocytes and determinate purityMethods The astrocytes were isolated from cerebral cortex of neonatal rats which are born within 3d, 1.5×107 count by inoculation with 0.01% in the poly-L-lysine-coated 75cm2 bottle, placed in the incubator (5% CO2, 95% O2, 37OC),cultivating by DMEM media added with 15% fetal calf serum and depurating by swinging and passaging , we will observe the whole of morphologic change, identify astrocytes by immunofluorescence staining. Results high purity astrocytes can be cultured by high serum-free culture and shaking ,and with the gradual passage, the cell will become more purified gradually , the morphologic of astrocytes also gradual chang with passage, the cell turn on positive for GFAP staining, normal astrocytes lanes triangular cells, membrane smooth, clear boundary, and the number of synapses connected long, GFAP-positive cells accounted for proportion of the total cell count more than 95%.Conclusion newborn Rat brain can make primary astrocytes ,and high-purity astrocytes can be btained by shaking and culture high serum medium.The second part The establishment of Primary astrocytes injury modelObjectives To establish the primary culture and nulli-nutrition model of astrocytes, to observe morphologic change. Methods The astrocytes were isolated from cerebral cortex of neonatal rats which are born within 3d, 1.5×107 count by inoculation with 0.01% in the poly-L-lysine-coated 75cm2 bottle, placed in the incubator (5% CO2, 95% O2, 37OC),cultivating by DMEM media added with 15% fetal calf serum and depurating by swinging and passaging, through PBS buffer instead of the medium for 3h making nulli-nutrition model, to observe cell morphological changes before and after Lacking of nutrition. Results High serum-free culture through the shaker will be of high purity astrocytes, and with gradual passage, can be obtained more purified astrocytes, after lacking of nutrition for 3h ,part of cells'body become smaller cell, synapse become shorter and connections between cells reducing or disappear, few of cells become round, after re-nutrition, the majority of cell body become larger, and the synapse become longer and intertwined, individual cell death. Conclusions Shaking availably after DMEM media with high-serum can obtain high-purity astrocytes, primary astrocytes may tolerance a certain lack of nutritional damage, the restoration of nutrition can be restored to normal form.The third part The experimental research on Methylprednisolone and erythropoietin in combination on astrocytes in vitroObjective To Study of methylprednisolone (MPSS) and erythropoietin (EPO) alone and combination in vitro primary role of astrocytesMethods The astrocytes were isolated from cerebral cortex of neonatal rats which are born within three days , cultivating by DMEM media added with 15% fetal calf serum and depurating by swinging and passaging, Experiment is divided into normal group, the normal + MPSS group, the normal + EPO group, the normal + MPSS + EPO group, injury group, + MPSS group damage, injury + EPO group, injury + MPSS + EPO group。to simulate nulli-nutrition model by substituting medium for PBS buffer solution 3h then applying MPSS,EPO,MPSS and EPO and rec-nutrition immediately ,we will go on culturing for three days, verificating astrocytes by immunofluorescent technique; detecting activity of astrocytes; determining GFAP of change by PCR.Results hypso-purity(95%)astrocytes can be acquired and more depurative by passaging,part of astrocytes will be dying after nulli-nutrition ;three days after applying MPSS ( 10umol/l ),EPO(10umol/l),MPSS(10umol/l)and EPO(10umol/l)into each group respectively, both cytoactive and GFAP expression were increased greatly in the damaged group ,and in MPSS and EPO with contrast to MPSS ,EPO lonely;while all the thing are opposite for normal astrocytes.Conclusions Appropriate dose of MPSS with EPO on the lack of nutrition combined injury astrocytes have a significant protective effect, and with the separate application of MPSS, EPO has significant compared to the role, while the MPSS, EPO alone, MPSS and EPO Application of United of normal astrocytes there was no significant protective effect.