【作者】 于宝国；

【导师】 张永亮；

【作者基本信息】 河北医科大学 ， 病理学与病理生理学， 2008， 硕士

【摘要】 目的:建立原代培养星形胶质细胞液压冲击损伤模型,利用双向电泳结合质谱技术观察脑皮质星形胶质细胞液压冲击损伤后蛋白质组的表达变化,探寻脑损伤的特异性生物标志蛋白。方法:取出生后24h内SD大鼠(军事医学科学院实验动物中心提供)大脑皮质组织,于含有10%胎牛血清的DMEM-F12培养基中进行星形胶质细胞体外分离培养和纯化,并以胶质纤维酸性蛋白(GFAP)免疫细胞化学染色进行纯度鉴定。随机将细胞分为对照组和损伤后4h、8h、12h、24h、48h组,复制Scott’s细胞液压冲击损伤模型(0.2MPa)。用台盼蓝染色观察星形胶质细胞损伤前后形态学变化,测培养液中LDH活力反映细胞的损伤程度,PI/Hoechst33342染色进行凋亡分析。提取细胞总蛋白,经双向电泳和MALDL-TOF质谱分析鉴定差异蛋白质。结果:1星形胶质细胞原代培养和纯化:经2-3次摇床传代纯化培养后的星形胶质细胞形态均一,胞体较大而扁、胞质较丰富、形状不规则;胞突丰富、分支多呈放射状,一级胞突较粗,常有二级分支;胞核圆形或椭圆形,常偏于胞体一侧。培养至24-26d,细胞生长状态良好,铺满皿底,形成大致均一的扁平细胞层。2星形胶质细胞的纯度鉴定:采用GFAP免疫细胞化学染色(SP法),计数结果显示培养的星形胶质细胞纯度可达94.9%±1.9%。3液压冲击损伤后星形胶质细胞形态学的改变:光镜下观察,细胞随时间发生间隙增大、肿胀、变圆、恢复,部分细胞发生皱缩、胞膜破裂、脱壁等显著的形态学改变;PI/Hoechst33342(1:1)荧光双染可见凋亡细胞。4液压冲击损伤后细胞培养液中LDH的变化:与正常对照组细胞培养液相比,损伤后各组细胞培养液中LDH活力均降低,4h组降至最低,与对照组相比有统计学差异(P<0.05)。然后其它组逐渐上升,48h组与对照组相比无统计学差异(P>0.05)。5液压冲击损伤后星形胶质细胞双向电泳图谱分析:双向电泳图谱经PDquest软件分析检测到的各组总蛋白点数:对照组1369±56,4h组1325±94,8h组1136±81,12h组1197±112,24h组1257±63,48h组1176±73。蛋白点多分布于pI5.2-6.6、MW30kDa-70kDa的范围;以对照组为参考胶,平均图谱匹配率达82%以上。损伤后共有265个蛋白质点的相对强度与正常对照组相比有显著性差异(P<0.05),其中38个蛋白质点在损伤后表达升高,111个蛋白质点在损伤后表达下降,45个蛋白质点为损伤后新表达,有71个蛋白质点在损伤后各组均未表达。6差异蛋白质的质谱鉴定:对9个差异蛋白点行MALDI-TOF质谱鉴定,其中8个点得分大于61分(P<0.05),分别为60S酸性核糖体蛋白P2(60S acidic ribosomal protein P2)、细胞视黄醇结合蛋白(cellular retinol binding protein, Crbp)、脑脂肪酸结合蛋白7(brain fatty acid binding protein 7, FABP7)、S-100钙结合蛋白A11(S100 calcium binding protein A11, S100A11 )、假设蛋白LOC685814 ( hypothetical protein LOC685814)、真核生物翻译起始因子1A(Eukaryotic translation initiation factor 1A, eIF-1A)、乳腺癌扩增序列2同源体( Breast carcinoma amplified sequence 2 homolog, BCAS2)、钙结合蛋白3(calponin 3)。其中对FABP7行Western-blot验证,结果显示:FABP7在星形胶质细胞中存在表达,而且该蛋白在星形胶质细胞液压冲击损伤后的变化趋势与双向电泳图像经PDQuest软件分析后得到的蛋白质变化趋势相一致。结论:1液压冲击损伤可引起星形胶质细胞形态学发生改变,且有一定的时间依从性。2液压冲击损伤能导致星形胶质细胞蛋白质组表达发生变化,损伤后共有265个蛋白点的相对强度与正常对照组相比有显著性差异,对其深入研究,有可能探寻到脑损伤的生物标志蛋白。3经MALDL-TOF质谱分析初步确定的8种蛋白大致分为代谢调节、信号转导调节、翻译起始相关类及其它等四类,这些蛋白表达的变化表明它们在参与星形胶质细胞损伤后早期反应、促进细胞功能恢复、抑制细胞继发性损伤、参与细胞骨架组织重组及改善蛋白质合成等环节起重要作用,对其进行进一步研究,有望深入了解脑损伤的分子机制。更多还原

【Abstract】 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.更多还原