原代培养大鼠脑星形胶质细胞液压冲击损伤后蛋白差异性表达的研究
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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。
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.
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。
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.
引文
1 Kobeissy FH, Ottens AK, Zhang ZQ, et al. Novel Differential Neuroproteomics Analysis of Traumatic Brain Injury in Rats. Molecular & Cellular Proteomics, 2006, 5: 1887~1898
2 Raivich G. Neuroglial activation repertoire in the injured brain: graded responsive mechanisms and cues to physiological function. Brain Res. Brain Res. Rev, 1999, 30: 77~105
3 Bovolenta P, Wandosell F, Nieto GM, et al. CNS glial scar tissue: a source of molecules which inhibit central neurite outgrowth. Prog Brain Res, 1992, 94: 367~379
4 何家全,蔡文琴,杨忠,等.大鼠中枢神经系统大胶质细胞对神经元轴突生长影响的离体实验观察.第三军医大学学报, 1999, 21(9): 630~633
5 薛庆善,郭畹华.大鼠大脑皮质星形胶质细胞的体外培养及其促神经突起生长作用研究.神经解剖学杂志,1996, 12(2): 151~155
6 薛庆善,肖渝平.神经胶质细胞的体外培养方法.解剖科学进展,1999, 5(2): 113~117
7 McCarthy KD, Vellis JD. Preparation of separate astroglial and oligodendroglial cell culture from rat cerebral tissue. The Journal of Cell Biology, 1980, 85: 890~902
8 Hildebrand B,Olenik C,Meyer DK. Neurons are generated in confluent astroglial cultures of rat neonatal neocortex.Neuroscience, 1997, 78(4): 957~966
9 Scott R, Shepard MD, Jamshid BG, et al. Fluid percussion barotrauma chamber: a new in vitro model for traumatic brain injury. J Surgical Res, 1991, 51(5): 417~424
10 陈伟杰, 张夔鸣, 杜显刚, 等. 大鼠脑液压冲击伤后细胞色素 C 表达的实验研究. 法律与医学杂志, 2004, 11(3): 219~221
11 Zhang XT, Liu EZ, Liu XQ, et al. Changes in and effective factors of microtubule-associated protein 2 in traumatic neurons. Chinese Medical Journal, 2001, 114(10): 1035~ 1038
12 周洪语,路丽明,沈建康. SD 大鼠星形胶质细胞的原代培养.中华神经医学杂志, 2003, 5(2): 346~355
13 Raivich G. Neuroglial activation repertoire in the injured brain: graded responsive mechanisms and cues to physiological function. Brain Res Brain Res Rev, 1999, 30(1): 77~105
14 Audouy S, Mallet J, Privat A, et al. Adenovirus-mediated suicide gene therapy in an in vitro model of reactive gliosis. Glia, 1999, 25(3): 293~303
15 Barbin G, Selak I, Manthorpe M, et al. Use of central neuronal cultures for the detection of neuronotrophic agents. Neuroscience, 1984, 12(1): 33~43
16 周洪语,路丽明,沈建康. SD 大鼠星形胶质细胞的原代培养.中华神经医学杂志,2003, 5(2): 346~355
17 Murphy EJ, Horrocks LA. A model for compression trauma:pressure-induced injury in cell cultures. J Neurotrauma, 1993, 10(4): 431~444
18 Zhang YH, Zhao N, Yi SY, et al. Injury model of central neuron by air pressure in vitro. J Fourth Mil Med Univ, 2002, 23(5): 423~425
19 Kinney JS, Willoughby KA, Liang S, et al. Stretch-induced injury of cultured neuronal, glial, and endothelial cells. Stroke, 1996, 27: 934~940
20 Ellis EF, Mckinney JS, Willoughby KA, et al. A new model for rapid stretch-induced injury of cells in culture: Characterization of the model using astrocytes. J Neurotrauma, 1995, 12(3): 325~339
21 Faden AI, O'Leary DM, Fan L, et al. Selective blockade of the mGluR1 receptor reduces traumatic neuronal injury in vitro and improves outcome after trauma. Exp Neurol, 2001, 167(2): 435~444
22 Scott R, Shepard MD, Jamshzd BG, et al. Fluid percussion barotrauma chamber: a new in vitro model for traumatic brain injury. J Surg Res, 1991, 51: 417~424
23 Posmantur RM, Zhao X, Kampfl A, et al. Immunoblot analyses of the relative contributions of cysteine and aspartic proteases to neurofilament breakdown products following experimental brain injury in rats. Neurochem Res, 1998, 23(10): 1265~1276
24 袁琼兰,李瑞祥,羊惠君,等. 大鼠脑缺血再灌流时神经元损伤与星形胶质细胞的反应.神经解剖学杂志,2000,16(3): 242~246
25 赵从建,贾宇峰,丁勤学等.中枢神经蛋白质组分析中双向电泳技术的建立.生物化学与生物物理进展,2000, 27 (6): 645~650
26 Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem, 1976, 72: 248~254
27 Klose J, Kobalz U. Two-dimensional electrophoresis of proteins: An undated protocol and implications for a functional analysis of the genome. Electrophoresis, 1995, 16: 1034~1059
28 Wilkins MR, Sanchez JC, Gooley AA, et al. Progress with proteome projects: why all proteins expressed by a genome should be identified and how to do it. Biotechnol Genet Eng Rev, 1996, 13: 19~50
29 赵从建,郭尧君,刘少君.蛋白质组分析的开门技术-双向电泳.现代科学仪器,2000, 5: 16~19
30 Rabilloud T. Two-dimensional gel electrophoresis in proteomics: Old, old fashioned, but it still climbs up the mountains. Proteomics, 2002, 2(1): 3~10
31 Fey SJ, Larsen PM. 2D or not 2D. Current Opinion in chemical biology, 2001, 5(1): 26~33
32 Gorg A, Postel W, Friedrich C, et al. Temperature -dependent spot positional variability in two-dimensional polypeptide patterns. Electrophoresis, 1991, 12(9): 653~658
33 Veerkamp JH, Maatman RG. Cytoplasmic fatty acid- binding protein their structure and genes. Prog lipid Res, 1995, 34(1): 17~52
34 Schroeder F, Jolly CA, Cho TH, et al. Fatty acid binding protein information: structure and function. Chemistry and Physics of Lipids, 1998, 92: 1~25
35 Van Nieuwenhoven FA, Van der Vusse GJ, Glatz JF. Membrane-associated and cytoplasmic fatty acid-binding proteins. Lipids, 1996, 31(1): 223~227
36 Anthony TE, Mason HA, Gridley T, et al. Brain lipid-binding protein is a direct target of Notch signaling in radial glial cells. GENES & DEVELOPMENT, 2005, 19: 1028~1033
37 王儒鹏.维甲酸信号传递系统在癌化学预防中的作用. 第三军医大学学报, 1999, 21(8): 42~44
38 陈惠平,张余光,张涤生.创伤后瘢痕的临床评估及综合治疗.整形再造外科杂志,2004, 1: 113~117
39 Guo XH, Chu MX, Zhou ZX. Molecular Biology of the retinol-binding proteins and their genes. HEREDITAS, 2004, 26(2): 257~262
40 Lei F, Hatten ME, Heintz N. Brain lipid-binding protein (BLBP): A novel signaling system in the developing mammalian CNS. Neuron, 1994, 12: 895~ 908
41 俞梦越,高润霖等.大鼠心肌梗死修复中肌纤维母细胞细胞视黄醇结合蛋白-1 的表达.中国循环杂志,2007, 02: 88~92
42 Ali MS, Harmer M, Vaughan R. Serum S100 protein as a marker of cerebral damage during cardiac surgery. British Journal of Anaesthesia, 2000, 85(2): 287~298
43 Michetti F, Gazzolo D. S100B protein in biological fluids: a tool for perinatal medicine. Clin Chem, 2002, 48(12): 2097~2104
44 Zimmer DB, Cornwall EH, Landar A, et al. The S100 protein family: history, function, and expression. Brain Res Bull, 1995, 37(4): 417~429
45 Zhao XQ, Naka M, Muneyuki M, et al. Ca(2+)-dependent inhibition of actin-activated myosin ATPase activity by S100C (S100A11), a novel member of the S100 protein family. Biochem Biophys Res Commun, 2000, 267~277
46 Arcuri C, Giambanco I, Bianchi R, et al. Subcellular localization of S100A11 (S100C, calgizzarin) in developing and adult avian skeletal muscles. Biochim Biophys Acta, 2002, 1600(1): 84~89
47 Kondo A, Sakaguchi M, Makino E, et al. Localization of S100C immunore2 activity in various human tissues. Acta Med Okayama, 2002, 56(1): 31~36
48 Sakaguchi M, Miyazaki M, Inoue Y, et al. Relationship between contact inhibition and intranuclear S100C of normal human fibroblasts. J Cell Biol, 2000, 149(6): 1193~1206
49 Sakaguchi M, Tsuji T, Inoue Y et al. Loss of nuclear localization of the S100C protein in immortalized humanfibroblasts. Radiat Res, 2001, 155: 208~214
50 Fattoum A, Roustan C, Smyczynski C, et a1. Mapping the microtubule binding regions of calponin. Biochemistry, 2003, 42(5): 1274~1282
51 Haag J, Aigner T. Identification of calponin 3 as a novel Smad-binding modulator of BMP signaling expressed in cartilage. Exp Cell Res, 2007, 313(16): 3386~3394
52 Abramczyk D, Tchórzewski M, Grankowski N. Non-AUG translation initiation of mRNA encoding acidic ribosomal P2A protein in Candida albicans. Yeast, 2003, 20(12): 1045~1052
53 Chaudhuri J, Si K, Maitra U. Function of Eukaryotic Translation Initiation Factor 1A (eIF1A) (Formerly Called eIF-4C) in Initiation of Protein Synthesis. J Biol Chem, 1997, 272(12): 7883~7891
1 Pandey A, Mann M. Proteomics to study genes and genomes. Nature, 2000, 405(6788): 837~846
2 Anderson NL, Anderson NG. Proteome and proteomics: new technologies, new concepts, and new words. Electrophoresis, 1998, 19(11): l853~l86l
3 Hillered L, Vespa PM, Hovda DA. Translational neuro- chemical research in acute human brain injury: the current status and potential future for cerebral microdialysis. J Neurotrauma, 2005, 22(1): 3~41
4 Wilkins MR, Sanchez JC, Gooley AA, et al. Progress with proteome projects: why all proteins expressed by a genome should be identified and how to do it. Biotechnol Genet Eng Rev, 1996, 13: 19~50
5 主初,梁宋平.肿瘤蛋白质组学.长沙:湖南科技出版社,2002: 1~7
6 秦晓冰,张敬川.蛋白质组学在乳腺癌中的研究与应用. 临床肿瘤学杂志,2006,11(5): 394~396
7 Yanagida M. Functional proteomics; current achievements. J Chromatogr B Analvt Technol Biomed Life Sci, 2002, 771: 89~106
8 Gorg A, Obermaier C, Boguth G, et a1. The current state of two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis, 2000, 21(6): 1037~1053
9 García-Campa?a AM, Gámiz-Gracia L, Baeyens WR, et al. Derivatization of biomolecules for chemiluminescent detection in capi11ary electrophoresis. J Chromatogr B Analyt Technol Biomed Life Sci, 2003, 793(1): 49~74
10 Ottens AK, Kobeissy FH, Golden EC, et al. Neuro- proteomics in neurotrauma. Mass Spectrom Rev, 2006,25(3): 380~408
11 Denslow N, Michel ME, Temple MD, et al. Application of proteomics technology to the field of neurotrauma, J Neurotrauma. 2003, 20(5): 401~407
12 Langen H, Bemdt P, Roder D, et a1. Two-dimensional map of human brain proteins. E1ectrophoresis, 1999, 20: 907~ 916
13 Yu LR, Conrads TP, Uo T, et a1. Global analysis of the cortical neuron proteome. Mol Cell Proteomics, 2004, 3(9): 896~907
14 Edgar PF, Douglas JE, Knight C, et a1. Proteome map of the human hippocampus. Hippocampus, 1999, 9(6): 644~650
15 Chen W, Ji J, Xu X, et a1. Proteomic comparison between human young and old brains by two-dimensional gel electrophoresis and identification of proteins. Int J Dev Neurosci, 2003, 21(4): 209~216
16 周波,杨伟,张景华,等.人脑枕叶区衰老进程的比较蛋白质组学研究.高等学校化学学报,2003, 24: 2202~2207
17 Davidsson P, Puchades M, Blennow K. Identification of synaptic vesicle,pre- and postsynaptic proteins in human cerebrospinal fluid using liquid-phase isoelectric focusing. Electrophoresis, 1999, 20(3): 431~437
18 Gauss C, Kalkum M, Lowe M, et a1. Analysis of the mouse proteome. (I) Brain proteins: separation by two-dimensional electrophoresis and identification by mass spectrometry and genetic variation. Eletrophoresis, 1999, 20(3): 575~600
19 Fountoulakis M, Schuller E, Hardmeier R, et a1. Rat brain proteins: two-dimensional protein database and variations in the expression leve1. Electrophoresis, 1999, 20(18): 3572~3579
20 Tsugita A, Kawakami T, Uchida T, et a1. Proteome analysis of mouse brain: two-dimensional electrophoresis profiles of tissue proteins during the course of aging. Electrophoresis, 2000, 21(9): 1853~1871
21 Langen H, Berndt P, Roder D, et a1. Two dimensional map of human brain proteins. Electrophoresis, 1999, 20: 907~916
22 Jenkins LW, Peters GW, Dixon CE, et al. Conventional and functional proteomics using large format two-dimensional gel electrophoresis 24 hours after controlled cortical impact in postnatal day 17 rats. J Neurotrauma, 2002, 19(6): 715~740
23 Conti A, Sanchez-Ruiz Y, Bachi A, et al. Proteome study of human cerebrospinal fluid following traumatic brain injury indicates fibrin(ogen) degradation products as trauma- associated markers. J Neurotrauma, 2004, 21(7): 854~863
24 Steiner S, Anderson NL. Pharmaceutical proteomics. Ann N Y Acad Sci, 2000, 9l9: 48~51
25 Santoni V, Moloy M, Rabiloud T. Membrane proteins and proteomics: unamour impossible. Electrophoresis, 2000, 21: 1054~1070
26 Lubec G, Krapfenbauer K, Fountoulakis M. Proteomics in brain research: potentials and limitations. Prog Neurobiol, 2003, 69(3): 193~211
27 Fields S. Proteomics in Genomeland. Science, 200l, 291 (5507): 1221~1224
2 Raivich G. Neuroglial activation repertoire in the injured brain: graded responsive mechanisms and cues to physiological function. Brain Res. Brain Res. Rev, 1999, 30: 77~105
3 Bovolenta P, Wandosell F, Nieto GM, et al. CNS glial scar tissue: a source of molecules which inhibit central neurite outgrowth. Prog Brain Res, 1992, 94: 367~379
4 何家全,蔡文琴,杨忠,等.大鼠中枢神经系统大胶质细胞对神经元轴突生长影响的离体实验观察.第三军医大学学报, 1999, 21(9): 630~633
5 薛庆善,郭畹华.大鼠大脑皮质星形胶质细胞的体外培养及其促神经突起生长作用研究.神经解剖学杂志,1996, 12(2): 151~155
6 薛庆善,肖渝平.神经胶质细胞的体外培养方法.解剖科学进展,1999, 5(2): 113~117
7 McCarthy KD, Vellis JD. Preparation of separate astroglial and oligodendroglial cell culture from rat cerebral tissue. The Journal of Cell Biology, 1980, 85: 890~902
8 Hildebrand B,Olenik C,Meyer DK. Neurons are generated in confluent astroglial cultures of rat neonatal neocortex.Neuroscience, 1997, 78(4): 957~966
9 Scott R, Shepard MD, Jamshid BG, et al. Fluid percussion barotrauma chamber: a new in vitro model for traumatic brain injury. J Surgical Res, 1991, 51(5): 417~424
10 陈伟杰, 张夔鸣, 杜显刚, 等. 大鼠脑液压冲击伤后细胞色素 C 表达的实验研究. 法律与医学杂志, 2004, 11(3): 219~221
11 Zhang XT, Liu EZ, Liu XQ, et al. Changes in and effective factors of microtubule-associated protein 2 in traumatic neurons. Chinese Medical Journal, 2001, 114(10): 1035~ 1038
12 周洪语,路丽明,沈建康. SD 大鼠星形胶质细胞的原代培养.中华神经医学杂志, 2003, 5(2): 346~355
13 Raivich G. Neuroglial activation repertoire in the injured brain: graded responsive mechanisms and cues to physiological function. Brain Res Brain Res Rev, 1999, 30(1): 77~105
14 Audouy S, Mallet J, Privat A, et al. Adenovirus-mediated suicide gene therapy in an in vitro model of reactive gliosis. Glia, 1999, 25(3): 293~303
15 Barbin G, Selak I, Manthorpe M, et al. Use of central neuronal cultures for the detection of neuronotrophic agents. Neuroscience, 1984, 12(1): 33~43
16 周洪语,路丽明,沈建康. SD 大鼠星形胶质细胞的原代培养.中华神经医学杂志,2003, 5(2): 346~355
17 Murphy EJ, Horrocks LA. A model for compression trauma:pressure-induced injury in cell cultures. J Neurotrauma, 1993, 10(4): 431~444
18 Zhang YH, Zhao N, Yi SY, et al. Injury model of central neuron by air pressure in vitro. J Fourth Mil Med Univ, 2002, 23(5): 423~425
19 Kinney JS, Willoughby KA, Liang S, et al. Stretch-induced injury of cultured neuronal, glial, and endothelial cells. Stroke, 1996, 27: 934~940
20 Ellis EF, Mckinney JS, Willoughby KA, et al. A new model for rapid stretch-induced injury of cells in culture: Characterization of the model using astrocytes. J Neurotrauma, 1995, 12(3): 325~339
21 Faden AI, O'Leary DM, Fan L, et al. Selective blockade of the mGluR1 receptor reduces traumatic neuronal injury in vitro and improves outcome after trauma. Exp Neurol, 2001, 167(2): 435~444
22 Scott R, Shepard MD, Jamshzd BG, et al. Fluid percussion barotrauma chamber: a new in vitro model for traumatic brain injury. J Surg Res, 1991, 51: 417~424
23 Posmantur RM, Zhao X, Kampfl A, et al. Immunoblot analyses of the relative contributions of cysteine and aspartic proteases to neurofilament breakdown products following experimental brain injury in rats. Neurochem Res, 1998, 23(10): 1265~1276
24 袁琼兰,李瑞祥,羊惠君,等. 大鼠脑缺血再灌流时神经元损伤与星形胶质细胞的反应.神经解剖学杂志,2000,16(3): 242~246
25 赵从建,贾宇峰,丁勤学等.中枢神经蛋白质组分析中双向电泳技术的建立.生物化学与生物物理进展,2000, 27 (6): 645~650
26 Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem, 1976, 72: 248~254
27 Klose J, Kobalz U. Two-dimensional electrophoresis of proteins: An undated protocol and implications for a functional analysis of the genome. Electrophoresis, 1995, 16: 1034~1059
28 Wilkins MR, Sanchez JC, Gooley AA, et al. Progress with proteome projects: why all proteins expressed by a genome should be identified and how to do it. Biotechnol Genet Eng Rev, 1996, 13: 19~50
29 赵从建,郭尧君,刘少君.蛋白质组分析的开门技术-双向电泳.现代科学仪器,2000, 5: 16~19
30 Rabilloud T. Two-dimensional gel electrophoresis in proteomics: Old, old fashioned, but it still climbs up the mountains. Proteomics, 2002, 2(1): 3~10
31 Fey SJ, Larsen PM. 2D or not 2D. Current Opinion in chemical biology, 2001, 5(1): 26~33
32 Gorg A, Postel W, Friedrich C, et al. Temperature -dependent spot positional variability in two-dimensional polypeptide patterns. Electrophoresis, 1991, 12(9): 653~658
33 Veerkamp JH, Maatman RG. Cytoplasmic fatty acid- binding protein their structure and genes. Prog lipid Res, 1995, 34(1): 17~52
34 Schroeder F, Jolly CA, Cho TH, et al. Fatty acid binding protein information: structure and function. Chemistry and Physics of Lipids, 1998, 92: 1~25
35 Van Nieuwenhoven FA, Van der Vusse GJ, Glatz JF. Membrane-associated and cytoplasmic fatty acid-binding proteins. Lipids, 1996, 31(1): 223~227
36 Anthony TE, Mason HA, Gridley T, et al. Brain lipid-binding protein is a direct target of Notch signaling in radial glial cells. GENES & DEVELOPMENT, 2005, 19: 1028~1033
37 王儒鹏.维甲酸信号传递系统在癌化学预防中的作用. 第三军医大学学报, 1999, 21(8): 42~44
38 陈惠平,张余光,张涤生.创伤后瘢痕的临床评估及综合治疗.整形再造外科杂志,2004, 1: 113~117
39 Guo XH, Chu MX, Zhou ZX. Molecular Biology of the retinol-binding proteins and their genes. HEREDITAS, 2004, 26(2): 257~262
40 Lei F, Hatten ME, Heintz N. Brain lipid-binding protein (BLBP): A novel signaling system in the developing mammalian CNS. Neuron, 1994, 12: 895~ 908
41 俞梦越,高润霖等.大鼠心肌梗死修复中肌纤维母细胞细胞视黄醇结合蛋白-1 的表达.中国循环杂志,2007, 02: 88~92
42 Ali MS, Harmer M, Vaughan R. Serum S100 protein as a marker of cerebral damage during cardiac surgery. British Journal of Anaesthesia, 2000, 85(2): 287~298
43 Michetti F, Gazzolo D. S100B protein in biological fluids: a tool for perinatal medicine. Clin Chem, 2002, 48(12): 2097~2104
44 Zimmer DB, Cornwall EH, Landar A, et al. The S100 protein family: history, function, and expression. Brain Res Bull, 1995, 37(4): 417~429
45 Zhao XQ, Naka M, Muneyuki M, et al. Ca(2+)-dependent inhibition of actin-activated myosin ATPase activity by S100C (S100A11), a novel member of the S100 protein family. Biochem Biophys Res Commun, 2000, 267~277
46 Arcuri C, Giambanco I, Bianchi R, et al. Subcellular localization of S100A11 (S100C, calgizzarin) in developing and adult avian skeletal muscles. Biochim Biophys Acta, 2002, 1600(1): 84~89
47 Kondo A, Sakaguchi M, Makino E, et al. Localization of S100C immunore2 activity in various human tissues. Acta Med Okayama, 2002, 56(1): 31~36
48 Sakaguchi M, Miyazaki M, Inoue Y, et al. Relationship between contact inhibition and intranuclear S100C of normal human fibroblasts. J Cell Biol, 2000, 149(6): 1193~1206
49 Sakaguchi M, Tsuji T, Inoue Y et al. Loss of nuclear localization of the S100C protein in immortalized humanfibroblasts. Radiat Res, 2001, 155: 208~214
50 Fattoum A, Roustan C, Smyczynski C, et a1. Mapping the microtubule binding regions of calponin. Biochemistry, 2003, 42(5): 1274~1282
51 Haag J, Aigner T. Identification of calponin 3 as a novel Smad-binding modulator of BMP signaling expressed in cartilage. Exp Cell Res, 2007, 313(16): 3386~3394
52 Abramczyk D, Tchórzewski M, Grankowski N. Non-AUG translation initiation of mRNA encoding acidic ribosomal P2A protein in Candida albicans. Yeast, 2003, 20(12): 1045~1052
53 Chaudhuri J, Si K, Maitra U. Function of Eukaryotic Translation Initiation Factor 1A (eIF1A) (Formerly Called eIF-4C) in Initiation of Protein Synthesis. J Biol Chem, 1997, 272(12): 7883~7891
1 Pandey A, Mann M. Proteomics to study genes and genomes. Nature, 2000, 405(6788): 837~846
2 Anderson NL, Anderson NG. Proteome and proteomics: new technologies, new concepts, and new words. Electrophoresis, 1998, 19(11): l853~l86l
3 Hillered L, Vespa PM, Hovda DA. Translational neuro- chemical research in acute human brain injury: the current status and potential future for cerebral microdialysis. J Neurotrauma, 2005, 22(1): 3~41
4 Wilkins MR, Sanchez JC, Gooley AA, et al. Progress with proteome projects: why all proteins expressed by a genome should be identified and how to do it. Biotechnol Genet Eng Rev, 1996, 13: 19~50
5 主初,梁宋平.肿瘤蛋白质组学.长沙:湖南科技出版社,2002: 1~7
6 秦晓冰,张敬川.蛋白质组学在乳腺癌中的研究与应用. 临床肿瘤学杂志,2006,11(5): 394~396
7 Yanagida M. Functional proteomics; current achievements. J Chromatogr B Analvt Technol Biomed Life Sci, 2002, 771: 89~106
8 Gorg A, Obermaier C, Boguth G, et a1. The current state of two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis, 2000, 21(6): 1037~1053
9 García-Campa?a AM, Gámiz-Gracia L, Baeyens WR, et al. Derivatization of biomolecules for chemiluminescent detection in capi11ary electrophoresis. J Chromatogr B Analyt Technol Biomed Life Sci, 2003, 793(1): 49~74
10 Ottens AK, Kobeissy FH, Golden EC, et al. Neuro- proteomics in neurotrauma. Mass Spectrom Rev, 2006,25(3): 380~408
11 Denslow N, Michel ME, Temple MD, et al. Application of proteomics technology to the field of neurotrauma, J Neurotrauma. 2003, 20(5): 401~407
12 Langen H, Bemdt P, Roder D, et a1. Two-dimensional map of human brain proteins. E1ectrophoresis, 1999, 20: 907~ 916
13 Yu LR, Conrads TP, Uo T, et a1. Global analysis of the cortical neuron proteome. Mol Cell Proteomics, 2004, 3(9): 896~907
14 Edgar PF, Douglas JE, Knight C, et a1. Proteome map of the human hippocampus. Hippocampus, 1999, 9(6): 644~650
15 Chen W, Ji J, Xu X, et a1. Proteomic comparison between human young and old brains by two-dimensional gel electrophoresis and identification of proteins. Int J Dev Neurosci, 2003, 21(4): 209~216
16 周波,杨伟,张景华,等.人脑枕叶区衰老进程的比较蛋白质组学研究.高等学校化学学报,2003, 24: 2202~2207
17 Davidsson P, Puchades M, Blennow K. Identification of synaptic vesicle,pre- and postsynaptic proteins in human cerebrospinal fluid using liquid-phase isoelectric focusing. Electrophoresis, 1999, 20(3): 431~437
18 Gauss C, Kalkum M, Lowe M, et a1. Analysis of the mouse proteome. (I) Brain proteins: separation by two-dimensional electrophoresis and identification by mass spectrometry and genetic variation. Eletrophoresis, 1999, 20(3): 575~600
19 Fountoulakis M, Schuller E, Hardmeier R, et a1. Rat brain proteins: two-dimensional protein database and variations in the expression leve1. Electrophoresis, 1999, 20(18): 3572~3579
20 Tsugita A, Kawakami T, Uchida T, et a1. Proteome analysis of mouse brain: two-dimensional electrophoresis profiles of tissue proteins during the course of aging. Electrophoresis, 2000, 21(9): 1853~1871
21 Langen H, Berndt P, Roder D, et a1. Two dimensional map of human brain proteins. Electrophoresis, 1999, 20: 907~916
22 Jenkins LW, Peters GW, Dixon CE, et al. Conventional and functional proteomics using large format two-dimensional gel electrophoresis 24 hours after controlled cortical impact in postnatal day 17 rats. J Neurotrauma, 2002, 19(6): 715~740
23 Conti A, Sanchez-Ruiz Y, Bachi A, et al. Proteome study of human cerebrospinal fluid following traumatic brain injury indicates fibrin(ogen) degradation products as trauma- associated markers. J Neurotrauma, 2004, 21(7): 854~863
24 Steiner S, Anderson NL. Pharmaceutical proteomics. Ann N Y Acad Sci, 2000, 9l9: 48~51
25 Santoni V, Moloy M, Rabiloud T. Membrane proteins and proteomics: unamour impossible. Electrophoresis, 2000, 21: 1054~1070
26 Lubec G, Krapfenbauer K, Fountoulakis M. Proteomics in brain research: potentials and limitations. Prog Neurobiol, 2003, 69(3): 193~211
27 Fields S. Proteomics in Genomeland. Science, 200l, 291 (5507): 1221~1224