| Objective: To establish the model of fluid percussion injury of primary cultured astrocytes, and to observe the alteration of protein expression pattern in astrocytes in vitro following fluid percussion injury via two dimensional gel electrophoresis and mass-spectrometry (MS), and to find new biomarkers of brain injury .Methods: In this study, the cells suspension were prepared from cerebral cortex of 24 hours-old SD rats , then be cultured primarily in DMEM-F12 medium which included 10% FCS. The cells were identified by immunocytochemical staining with anti-GFAP and then were randomly divided into normal control group and post-injury 4h,8h,12h,24h and 48h groups which were subjected to Scott's fluid percussion injury. Extent of cell injury was qualitatively assesssed by trypan blue, PI/Hoechst33342 staining and LDH levels. The protein samples, extracted from astrocytes of different groups, were applied to 2D electrophoresis. Then the types and roles of the different expressed proteins were ascertained preliminarily by mass spectrometry.Results: 1 Isolation and purification of Primary cultured astrocytes: The purified astrocytes, which were obtained after 2 or 3 times subcultured, showed uniform appearance,big cell body, abundant cytoplasm, many longer apophyses and round or ovi-round karyons which leaned to one side. It coincided with the form of astrocytes. After cells were cultured 24 or 26 days, they almost spreaded all the bottom of the culture capsules and had a stable condition, and then they can be used to establish the model of fluid percussion injury.2 Identification of astrocytes: The purification of astrocytes was identified by immunocytochemical staining of glial fribrillary acidic protein (GFAP). It demonstrated that 94.9%±1.9% percents were positive in all the cells.3 Morphological study of astrocytes following fluid percussion injury: followed astrocytes moderate injury, the typical morphological features of injury cells were detected via Microscopy: intercellular space increasing, swollen, turning round; part of the cells shrinkage, cytomembrane ruptured and scattered; the apoptotic cells can be observed by PI-Hoechst fluorescence staining.4 The change of lactate dehydrogenase (LDH) in culturing solution following fluid percussion injury: Compared with the control group, the activities of LDH were reduced in each of the post-injury groups. Among the total, 4h group reached to the minimum, and had a statistical difference (P<0.05). Then the activities of 8h, 12h and 24h groups gradually increased, 48h group and the control group showed no significant difference (P>0.05).5 The results of 2-DE following fluid percussion injury of astrocytes: 1369±56,1325±94,1136±81,1197±112,1257±63,1176±73 protein spots were detected respectively in control,4h,8h,12h,24h and 48h groups via PDquest software. Furthermore, most of these spots were dispersed into the scopes of pI5.2-6.6 and MW 30kDa-70kDa;The image of control group was considered as the master image, the average match rate was more than 82%. Dynamic changes were identified and total 265 differe ntially expressed proteins were detected in this study from the 2DE gels (P<0.05), which included 38 up-regulated spots, 111 down-regulated spots, 45 new-increased spots and 71 protein-spots couldn't be detected in different period after injury.6 Identification of the different displayed protein spots via MALDI-TOF-MS: Nine protein spots were analyzed via MALDI-TOF-MS, however only eight of them were confirmed in the end, which scores were more than 61 (P<0.05). They are 60S acidic ribosomal protein P2, cellular retinol binding protein (Crbp), brain fatty acid binding protein 7, S100 calcium binding protein A11, hypothetical protein LOC685814, Eukaryotic translation initiation factor 1A (eIF-1A), Breast carcinoma amplified sequence 2 homolog and calponin 3. Brain fatty acid binding protein7 was authenticated via Western-blot. The result demonstrated that brain fatty acid binding protein 7 has expressed in astrocytes, and have the same tendency of protein change with the 2D images which were analyzed by PDQuest7.0 software.Conclusion: 1 The alteration of morphology in astrocytes could be induced after fluid percussion injury, and there is a certain amount of time compliance.2 Fluid percussion injury led to changes in protein expression patterns in astrocytes. There were significant differences in the relative strength of 265 protein points compared with the normal control group, so it is possible to explore the biomarker protein in brain injury in their further study.3 Eight kinds of protein, which were divided into regulating metabolism, signal transduction regulation, translation start-related and other four categories, were identified via MALDI-TOF-MS analysis the initially. These changes in protein expression indicated that they play important role in some aspects, for example in participation with early response after astrocytes injury, promotion of functional recovery, inhibition of secondary injury, reorganization of cytoskeleton and improvement of protein synthesis. The further study could be useful for molecular mechanism of brain injury.
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