| The spinal cord injury (SCI) is the serious trauma, and often results in high mutilation and death rate. With development of traffic and acceleration of industrialization, the patients suffered from SCI tend to increase yearly. Thus, the research on SCI has become the hottest field and is full with difficulties mainly because of the unclear molecular mechanism of SCI and the limited regeneration ability of spinal cord after lesion.Astrocyte, predominant glia cell in CNS, is one of the earliest cells respond to CNS lesion. The in-depth research on the changes and reactions of astrocyte to CNS injury and its theory has been carried out in recent years. However the molecular mechanism on the actions of astrocytes after injury is still unclear. The study on the response of astrocyte after SCI may redound to clarify the molecular mechanism of SCI.To seek the response and the dynamic changes in proteins expression of astrocytes after injury, the 2D electrophresis is employed to visualize the different expression proteins via a new design mechanical injury model in vitro.First, we isolated and purified astrocytes from rat spinal cord. The isolated spinal cord tissue from the newborn Wistar rats was cut into pieces with scissor and then digested with trypsin. And the whole cells of spinal cord were mixed to cultivate. Thenthe pured astrocytes, with over 97% purity identified with immunofluorescence, were separated by mechanical dissociation from adhesive layer cells, and re-seeded on culture dishes at a density about 3.0x104 /ml. After being cultured for another 12 days, the pured astrocytes in the stable state, shaped a thin and flat layer with short bulky processes, were applied for lesion experiments.It is crucial to build a lesion model of cultured astrocytes to investigate the changes after injury. To achieve this goal, a new-designed standard template with pre-designed lines according to the character of astrocytes with processes, was used in this experiment. The easy-controlled template was put under the culture dishes to reveal the cut matching to the cross-sectional lines on it by a sharp razor blade. And the obvious cut injury was observed under phase contrast microscopey. The cultured dishes were divided into four groups: 24 hour, 72 hour and 120 hours after injury, and normal cultured dishes as control. All cultured cells were harvested for the protein sample preparation in 2DE electrophoresis study.The proteins, extracted from cultured astrocytes with lysis solution via ultrasonic and ultracentrifu.gation, was stored at refrigerator under -75C and used for 2DE electrophoresis after quantitated by Bradford measurement method. 12 gels, with 3 gels per every group were used in this investigation.The parameter of the IPG as the first dimension electrophoresis condition was finally controlled at 30,000 Vhr according to the experiment, and the optimized formula of SDS PAGE gels were applied to the second dimension electrophoresis with other step such as fixed gel position that improved the comparability. The experiments showed that staining for 4 min with silver nitrate was the suitable condition for this sample as the key procedures.Those spots ranged from isoelectric point (pI) 3-10 and molecular weight (MW) 10-100 kDa were showed clearly in the protein expression 2DE maps. And the dynamic changes of proteins at the injury model after 24, 72 and 120 h were also analyzed.1785, 1806, 1809 and 1841 protein spots were detected respectively in control, 24 h, 72 h and 120 h via ImageMaster software. After the matching process had beenperformed both between and within groups, the unmatched spots were then considered as possible different proteins. And those spots should be comfirmed after the matching processure performed between groups. About 88% spots of experiment gels matched to those of reference gel in 24 h, which showed a high reproducibility.Three categories of dynamic changes were identified and total different 50 spots were detected in this study from the 2DE gels. One sort, with total 1...
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