| Retinal Muller cell is the most important glia type cell in the retina .It pierces through the whole layer of retina, supports and packs around the nerve cell, and plays an important role in the contusion of retina .Past evidence indicates that there was obvious pathological changes of Muller cells in the earlier period after the retina injury, but its occurrence mechanism is still unclear.The objective of this study was to establish a model of high air pressure wave traumatic injury in cultured ratinal Muller cell, which was based on a novel device that is capable of applying high pressure and high speed air, earlier period pathology changes after injury were observed,specially the differential expression of GFAP( Glia fibrillary acidic protein) and its relation with other pathologic changes. We used the model to observe the ultrastructure changes of injuryed cell, tested the expression of GFAP after injury by immunochemistry, analysized by computer image manipulation system, tested the change of LDH activity after injury by biochemistery , and detected cell apoptosis rate with in situ TENNEL method. This will help to understand the mechanism of early reactive changes of Muller cell after contusion of retina.The main result and conclusions are as follows:1. We established a new method for the purification culture of retinal Muller cell. Muller cell is an ideal type in vitro traumatic study that can get a purity above 97% by using the multiple attaching and changing culture medium again and again.2. We developed a device to make injury to the vitro cultured cell with high speed and pressure air. The inducing injury parameters can be controlled and on- line tested by the computer. The device has the advantages that it could be manipulated with ease and convenience, making the injury controllable and repeatable.3. We developed a new model of traumatic injury in cultured ratinal Muller cell, which is based on a novel device that is capable of applying high pressure and high speed air. There were few studies about in vitro traumatic model of retinal Muller cell in ourcountry .Our study showed that the degree of cell injury can be controlled by different gas pressure while the model can be taken as a primary traumatic model, which provide information about the changes of cell morphology and function induced by injury at cell and molecular level.4. We have systematically observed the earlier period pathologic changes after Miiller cell injury. The study shows in earlier period after cell injury, because of the injury to cell membrane, a large quantity of death cell showed trypan blue dyeing positive. With the pass of time, 3 hours after injury, at the effects of the secondary trauma, apoptosis mechanism was actuated, many celles showed apoptosis at this time.5. We systemically tested the expression of GFAP after injury by immunochemistry, analysized the result by computer image manipulation system. Significant increase in GFAP through immunolabelling of Muller cell was noted in injured cells compared with normal cells . The results show that increased expression of GFAP changes were observed 3h after injury. Then it maintained high level of expression in 24h, the results were related with injury degree .We proved that injury of the cell membrane is one of the factor to trigger the expression of GFAP. We also discovered that the expression of GFAP increased obviously while a large quantity of cell apoptosis 3h after injury, manifested that apoptosis was actuated at ahnost the same time. It may be explained that they were triggered by the same signal path. With apoptosis of Miiller cells, those surviving cell begin proliferate and become hypertrophy. |
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