The Effect Of Erythropoietin And Methylprednisolone In Combination On Astrocytes

The first part Culture and identification of astrocytes from ratsObjectives To make primary astrocytes from cerebral cortex of neonatal rat , to culture and passage for identification and determination of purity of astrocytes.Methods The astrocytes were isolated from cerebral cortex of neonatal rats which are born within 3 days, inoculated in the 0.01% poly-L-lysine-coated 75cm~2 bottle by 10~7~10~9 /L, placed in the incubator (5% CO~2, 37℃), cultivated by DMEM/F12 media added with 15% fetal bovine serum for 7-10 days, and depurated by differential velocity adherent technique, swinging and passaging , we will observe the morphological change of the cells, identify the astrocytes by immunofluorescence staining.Results High purity astrocytes can be achieved by combination of differential velocity adherent technique and shaking ,and with the gradual passage, the cell will become more purified gradually , the morphologic of astrocytes also chang gradually, the cell turn on positive for GFAP staining, normal astrocytes lanes triangular cells, membrane smooth, clear boundary, and cytoplasmic process long, GFAP-positive cells accounted for more than 95% of the total cell count.Conclusion Newborn rat brain cortex can make primary astrocytes ,and high-purity astrocytes can be obtained by shaking 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 morphological change.Methods The astrocytes were isolated from cerebral cortex of neonatal rats which are born within 3 days, inoculated in the 0.01% poly-L-lysine-coated 75cm~2 bottle by 107~109 /L, placed in the incubator (5% CO~2, 37℃), cultivated by DMEM/F12 media added with 15% fetal bovine serum for 8 days, and depurated by differential velocity adherent technique, swinging and passaging. Make nulli-nutrition model by PBS buffer instead of the medium for 3 h to observe cell morphological changes before and after lacking of nutrition.Results After lacking of nutrition for 3 h, part of cells'body become smaller, cytoplasmic process become shorter, few of cells become round, after re-nutrition, it was similar with the state befor lacking of nutrition, the majority of cell body become larger, the cytoplasmic process become longer and intertwined, few of cells dead.Conclusions Shaking and differential velocity adherent technique 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 effect of erythropoietin on astrocytesObjective To study the effect of erythropoietin (erythropoietin, EPO) on astrocytes (AST) in vitro culture.Method The astrocytes were isolated from cerebral cortex of neonatal rats which are born within three days, cultivating by DMEM/F12 media added with 15% fetal calf serum and depurating by swinging and passaging, to simulate spinal cord ischemia model by substituting medium for PBS buffer solution 3 h, then applying erythropoietin and rec-nutrition immediately, we will go on culturing for 0 h, 0.5 h, 1 h, 1.5 h, 2 h, 3 h, 6 h, 12 h, 18 h, verificating astrocytes by immunofluorescent technique; detecting activity of Ast by MTT; determining changes of AQP4 mRNA expression by PCR.Results Hypso-purity (95%) astrocytes can be acquired and more depurative by passaging, part of astrocytes will be dying after nulli-nutrition, the expression of AQP4 increased.Conclusions Half an hour of the using of EPO will have obviously effect for the AQP4 expression of dinjuried astrocytes.The fourth part Experimental research of erythropoietin and methylprednisolone in combination on astrocytesObjective: To study the effect of erythropoietin (EPO) and methylprednisolone (MPSS) in combination on Ischemic injuried astrocytes (AST) in vitro culture.Method The astrocytes were isolated from cerebral cortex of neonatal rats born within three days, cultivating by DMEM media added with 15% fetal calf serum and depurating by swinging and passaging, verificating astrocytes by immunofluorescent technique. To simulate ischemic model by substituting medium for PBS buffer solution 3h then applying EPO (10 u/ml), MPSS (10 ug/ml), EPO (10 u/ml) + MPSS (10 ug/ml), EPO (5 u/ml) + MPSS ( 5ug/ml)and DMEM media added with 15% fetal calf serum immediately, we will go on culturing, detecting activity of Ast by MTT, determining changes of AQP4 mRNA expression by PCR.Results Hypso-purity (95%) astrocytes can be acquired and more depurative by passaging, part of astrocytes will be dying after Ischemic, EPO and MPSS have obviously protection effect for damaged astrocytes .Conclusions The applying of half dose of EPO and MPSS have obviously protection effect for damaged astrocytes.