ABSTRACT
Objective To investigate the effect of ski gene in migration process of astrocytes in rats. Methods Astrocytes were obtained from rats' cerebral cortex and cultured in vitro. siRNA targeting ski gene and negative control sequences were prepared. The ski-siRNA group, siRNA negative control group and untreated group were set in this experiment. The specific siRNA targeting ski gene was transfected into astrocytes with Lipofectamine?RNAiMAX Reagent. Then the ski protein levels were determined with Western blotting. After transfec-tion, the changes in migration of astrocytes were measured with wound scratch assay and Transwell migration assay. Results Western blot-ting showed that the expression of ski protein was significantly lower in the ski-siRNA group than in the siRNA negative control group and untreated group (F=132.957, P47.197, P69.187, P<0.001). Conclusion Ski knocked down by siRNA could inhibit the migration ability of astrocytes. It is a reminding that ski may take part in the migration process of astrocytes, and moreover, ski may play an important role in the formation of glial scar.
ABSTRACT
AIM:To explore the time-dependent change of Ski protein expression in normal and activated astrocytes in rats.METHODS:The astrocytes were obtained from rat cerebral cortex and cultured in vitro.The astrocytes were treated with LPS and scratch injury for activation.Western blot analysis was used to determine glial fibrillary acidic protein (GFAP) and Ski protein levels in activated astrocytes at a series of time points.The indirect immunofluorescence staining method was performed to detect the location of Ski protein in the astrocytes.RESULTS:The protein of GFAP was naturally expressed in the astrocytes, beginning to increase after treated with LPS and scratch injury.Little protein expression of Ski in the normal astrocytes was observed.The Ski protein expression began to increase after treated with 1 mg/L LPS, peaked at 4 d (P<0.05) and then deceased, but was stills higher than that in the normal cells.The protein expression level of Ski after scratch injury was highly consistent with above mentioned.Ski was mainly observed in the nucleus of the normal cells and the cells treated with LPS for 6 d, while it was observed in the cytoplasm 2 and 4 d after treated with LPS.CONCLUSION:The protein of Ski is expressed in the astrocytes, and the expression level is increased in activated astrocytes,mainly located in the nucelus.Ski may plays an essential roles in the processes of activation and proliferation of astrocytes.
ABSTRACT
Objective To investigate the role of ski in proliferation of astrocytes and the molecular mechanisms in rats. Methods Astro-cytes were obtained from cerebral cortex of a three-day old rat and cultured in vitro. siRNA targeted to ski and negative control sequences were prepared. The astrocytes were divided into ski-siRNA group, siRNA negative control group and untreated control group, while the spe-cific siRNA targeting ski negative control sequences were transfected into astrocytes with Lipofectamine? RNAiMAX Reagent. The protein levels of ski, glial fibrillary acidic protein (GFAP) and Cyclin D1 were determined with Western blotting. The proliferation of astrocytes were measured with CCK8 assay. The cell-cycle of astrocytes were analyzed with flow cytometer. Results The protein level of ski (F=38.611, P30.507, P48.425, P<0.01), compared with the control groups. Conclusion ski knocking down by siRNA significantly inhibits the proliferation of astro-cytes, which may associate with the down-regulation of Cyclin D1 expression.
ABSTRACT
Objective To explore the expression and the changes of ski with time in the injured spinal cord in rats. Methods Sixty adult female Sprague-Dawley rats were randomly divided into sham group (n=30) and injury group (n=30), each group were further divided into 1 week, 2 weeks, 4 weeks, 8 weeks and 12 weeks subgroups, with 6 rats in each subgroup. Spinal cord injury at T10 was established with modi-fied Allen's technique (10 g × 25 mm) in the injury group. The hindlimbs behavior of rats was rated with Basso-Beattie-Bresnahan (BBB) scores 1 day, 3 days, 1 week, 2 weeks, 4 weeks, 8 weeks and 12 weeks after spinal cord injury. Three rats in each subgroup were stained with HE staining to observe the pathological changes of the spinal cord and the formation of cavity. The other 3 rats were analyzed with im-munofluorescence staining of ski and semi quantitative analysis. Results The BBB scores of each time point were less in the injury group than in the sham group (P<0.05). Necrosis was the major pathological change in the injury groups 1 and 2 weeks after injury;cystic cavity completely formed 4 weeks after injury, with dense scar tissue around it;there was no significant change in the cavity and scar 8 and 12 weeks after injury, however, the adjacent spinal cord was obviously thinner. Ski expressed little in the normal spinal cord, and expressed more and more after injury, peaked at 8 weeks and decreased then. Ski was mainly observed in white matter in the sham group and 12 weeks injury subgroup, which was in gray matter 2, 4 and 8 weeks after injury. Ski was highly expressed around the cavity in injury center and formed high expression band. Conclusion Ski expresses after spinal cord injury in rats, that may be associated with the activation and prolif-eration of astrocytes and the formation of glial scar.