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Objective To explore the expression and change of ski-interacting protein (SKIP) in rats after spinal cord injury. Methods A total of 60 adult female Sprague-Dawley rats were randomly divided into sham group (n=30) and spinal cord injury (SCI) group (n=30), each group was further divided into five time points including one day, three days, five days, seven days, and 14 days with six rats in each time points. The model was established at T10 with modified Allen's technique, and the sham group only bit the lamina of rats. The hindlimbs behavior was assessed with Basso-Beattie-Bresnahan (BBB) score at each time point. The pathological changes of spinal cord neurons were detected with Nissl staining. The expression of SKIP were observed with immunofluorescence staining. Results The BBB scores were signif-icantly lower in each time point in SCI group than in the sham group (t>48.267, P<0.001). Compared with the sham group, Nissl bodies in the cytoplasm of spinal cord neurons began to disintegrate, coalesce and irregularly distribute, the neurons began to degenerate and die on the fifth day, and the damage deteriorated on the 14th day. Immunofluorescence staining showed that SKIP expression was mainly expressed in the gray matter of the spinal cord and little expressed in the white matter. The expression of SKIP gradually increased after SCI, and reached a peak on the fifth day (t=-17.035, P<0.001) and decreased significantly on the 14th day (t=3.853, P<0.05). Conclusion SKIP may be a new signaling molecule, which play an important role in neuronal apoptosis after SCI.
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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.
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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.
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Wnt/β-catenin signaling pathway participates in cancer cell proliferation,invasion and metastasis and effectively induces drug resistance.It is also the key signal to mediate cancer carcinogenesis.Recent studies in vitro indicate that disturbance of Wnt/β-catenin signaling pathway can increase the sensitivity of the cancer cells to chemotherapeutic drugs.In-depth researches and analysis of tumor drug resistance induced by Wnt/β-catenin will provide potential targets and possible therapeutic means for the treatment of tumors.
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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.
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Ski, as an evolutionary conserved protein, is widely involoved in the proliferation and differentiation of many kinds of cells in different species. Ski also plays an irreplaceable role in many physiological and pathological processes of nervous system, including em-bryonic nervous system development, central and peripheral nervous system diseases, and so on, which may be assiciated with the signal pathways of transforming growth factor-beta and another family member bone morphogenetic protein.
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@#Objective To study the differentiation and proliferation ability of the spinal neural stem cells (NSCs) at different gestational ages in fetal rats. Methods Sprague-Dawley fetal rats were divided into group A (12 days of pregnancy), group B (14 days of pregnancy) and group C (16 days of pregnancy). NSCs were separated with enzyme-assisted microdissection. The diameter and numbers of NSCs balls were measured at different time. The cell growth curve was drawn with CCK8 colorimeter. NSCs were identified with BrdU/Nestin immunohistochemical staining. They were induced with 10% fetal bovine serum for 10 days, and the expression of β-tubulinⅢ and glial fibrillary acidic protein was detected with immunocytochemistry. Results There were cells expressed BrdU, Nestin, β-tubulinⅢ and GFAP in all the group. The most cells (22.74±0.79%) expressed β-tubulinⅢ in the group B, but no significant difference between group B and group C. The cell vitality on the 5th day of third-generation neural stem cells was the most in group B. Conclusion For enzyme-assisted microdissection, it may obtain more neurons to isolate the neural stem cells from 14 days of pregnancy pregnant rats.
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@#Objective To explore the learning and memory impairment and pathology in hippocampus in rats after spinal cord contu-sion. Methods 36 adult female Sprague-Dawley rats were randomly divided into sham group (n=18) and spinal cord injury group (n=18). Spinal cord injury model at T10 was established with modified Allen's technique (10 g × 25 mm). The hindlimbs behavior of rats was rated with Basso-Beattie-Bresnahan (BBB) scores once a week for 5 weeks. They were tested with motor evoked potentials (MEP) and Morris wa-ter maze 5 weeks after injury. The pathology of hippocampus was detected with HE staining 1 week, 3 weeks and 5 weeks after injury, 4 rats in a group, repectively. Results The BBB scores were significantly lower in the spinal cord injury group than in the sham group at each time point (P<0.05). The latencies of both N1 and P1 wave of MEP were significantly longer in the spinal cord injury group than in the sham group (P<0.001), while the amplitudes were significantly less (P<0.001). For the Morris water maze, the latency of arrival platform were sig-nificantly longer in the spinal cord injury group than in the sham group (P<0.001), and the time in target was significantly less (P<0.001), with more systematic positioning or annular positioning, while the sham group with more space-based positioning. Morphologically abnor-mal cells in hippocampus gradually increased since the first week after injury, with the decrease of cells survival, while it was normal in the sham group. Conclusion Spinal cord contusion can cause learning and memory impairment in rats, which may be related to injury in hippo-campus.
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Objective To study the differentiation and proliferation ability of the spinal neural stem cells (NSCs) at different gestational ages in fetal rats. Methods Sprague-Dawley fetal rats were divided into group A (12 days of pregnancy), group B (14 days of pregnancy) and group C (16 days of pregnancy). NSCs were separated with enzyme-assisted microdissection. The diameter and numbers of NSCs balls were measured at different time. The cell growth curve was drawn with CCK8 colorimeter. NSCs were identified with BrdU/Nestin immuno-histochemical staining. They were induced with 10%fetal bovine serum for 10 days, and the expression ofβ-tubulinⅢand glial fibrillary acidic protein was detected with immunocytochemistry. Results There were cells expressed BrdU, Nestin,β-tubulinⅢand GFAP in all the group. The most cells (22.74±0.79%) expressedβ-tubulinⅢin the group B, but no significant difference between group B and group C. The cell vitality on the 5th day of third-generation neural stem cells was the most in group B. Conclusion For enzyme-assisted microdissection, it may obtain more neurons to isolate the neural stem cells from 14 days of pregnancy pregnant rats.
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Zn. Conclusion Human activities developed along the Huaihe River does not influence the concentration of heavy metals in the sediment, no serious contamination of heavy metals in the sediment is found in the present investigation.