The Experimental Research Of Methylprednisolone On The Effects Of Expression Of Astrocyte AQP4 MRNA

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 75cm2 bottle by 107~109 /L, placed in the incubator (5% CO2, 370C), cultivated by DMEM/F12 media added with 15% fetal bovine serum for 8 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 nulli-nutrition 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 75cm2 bottle by 107~109 /L, placed in the incubator (5% CO2, 370C), 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 3h to observe cell morphological changes before and after lacking of nutrition.Using the RT-PCR detection the expression of GFAPmRNA response to nulli-nutrition in injuried astrocytes.Results After lacking of nutrition for 3h ,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. RT-PCR shows the expression of GFAPmRNA significantly increased in injuried astrocyte compared with control group,and there is statistical significance.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 experimental research of Methylprednisolone on the effects of expression of astrocyte AQP4 mRNA in vitroObjective To Study methylprednisolone (MPSS) on the change of expression of astrocyte AQP4 mRNA and on the mechanism of its effects on the edema of spinal cord.Methods SD rats'cerebral cortex astrocyte cells were removed three days after birth and cultured. Establish a lack-of-nutrition injured astrocyte model and treat with different doses of methylprednisolone. We used methyl thiazolyl tetrazolium (MTT) to detect cell activity , immunocytochemical method to detect the expression of AQP4, RT-PCR to detect the level of AQP4mRNA, and finally use the light microscope to observe the impact of lack of nutrition on morphology of injured astrocyte.Results In vitro, when astrocytes grew to the third generation and immersed in the PBS fluid for 3h ,then given DMEM/F12 medium with 10% fetal bovine serum for the 6-hour intervention of different MPSS concentrations ,we found the concentration of AQP4 mRNA expression levels were significantly lower than the control group (P <0.05), also with MPSS dose correlation.Conclusions MPSS demoted the AQP4 mRNA expression of astrocytes in vitro ,and AQP4 expression was dose-dependent with MPSS. Therefore, MPSS may inhibit AQP4 mRNA expression of astrocyte to alleviate the edema and depress the formation of glial scar in the central nervous system .